Registration Dossier

Administrative data

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 guideline study

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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Wash oil
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Substance type: organic
- Physical state: liquid
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic
- Age at study initiation: 8 - 10 wks
- Weight at study initiation: 18.1 - 21.9 g
- Housing: macrolon cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433
Concentration:
mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: emulsion, TS 30 %
- Irritation: no
- Lymph node proliferation response: not examined


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response (on primary proliferation of lymphocytes by incorporation of 3H-methyl thymidine)
Stimulation index (SI) is ≥ 3
Response increases in dose-related manner
Criteria for irritation potential: individual and mean ear weight


TREATMENT PREPARATION AND ADMINISTRATION:
Days 1-3: 25 µL test suspension in DAE 433 , 1x on day 1, 2, and 3
Days 4 and 5: no treatment
Day 6: Injection of 250 μL of phosphate-buffered saline (PBS) containing 7.5 x105 Bq (c. 21 µCi) of 3H-methyl thymidine
into all test and control mice via the tail vein. Five hours later, the animals were killed.



Positive control substance(s):
other: DNCB (dinitrochlorbenzene, 0.5% (w/v) solution)
Statistics:
Non-parametric Kruskal-Wallis test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all two-group comparisons [using software Statgraphic ® Centurion (version XV, USA)].

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Table 2 below: SI > 4 at >= 3 %
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Table 1 below

Any other information on results incl. tables

Table 1: Individual and mean radioactivity in cell suspensions (Report Table 4)

Activity (DPM, rounded)

Animal No.

NC

(1-5)

PC

(6-10)

30%

(11-15)

3%

(16-20)

0.3%

(21-25)

1

599

8066

2980

2659

1002

2

536

6900

4845

1995

852

3

795

6625

3250

2267

815

4

549

6332

3426

3283

856

5

527

6804

3171

2485

758

 Group

mean

601

6946

3534

2538

857

SI

1.00

11.55

5.88

4.22

1.42

NC = vehicle control; PC = positive control

Table 2: Individual and average ear weights (Report Table 5)

Weight of ear biopsies (milligrams)

Animal No.

NC

(1-6)

PC

(7-12)

30%

(13-18)

3%

(19-24)

0.3%

(25-30)

1

23.2

27.6

25.5

24.4

21.7

2

24.6

38.3

23.3

23.3

22.7

3

22.0

30.4

28.1

23.7

20.4

 4

23.2

35.5

25.8

23.8

21.3

5

22.9

29.5

26.6

24.0

21.8

 Group mean

23.18

32.26*

25.86*

23.86*

21.58

SD

0.93

4.16

1.75

0.37

0.83

NC = vehicle control; PC = positive control

* statistically significant with p =< 0.05, Mann-Whitney test

Table 3:  Summary table (Report Table 6)

Group

Radioisotope incorporation

in lymph nodes (rounded)

Ear weight

Mean DPM

SI

Mean (mg)

NC

601

1.0

23.18

PC

6946

11.55+

32.26*

30%

3534

5.88+

25.86*

3%

2538

4.22+

23.86

0.3%

857

1.42

21.58

Bold figures with + = values >= 3

NC = vehicle control; PC = positive control

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Wash oil considered to exhibit a weak sensitising potential, supported by the fact that a significant stimulatory effect was observed at a low dose (3 %)that failed to produce visible irritation.