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Description of key information

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 guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
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: ~ 19 - 23 g
- Housing: macrolon cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 46 – 70
- Photoperiod (hrs dark / hrs light): 12 / 12

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: suspension, TS 30 %
- Irritation: slight in 3 animals
- 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)].
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
SI > 4 at >= 3 % Experimental values: Concentration [%] / SI 0.3 / 1.65 3.0 / 4.67 30.0 / 13.03 EC3 was calculated by linear interpolation between concentrations of 0.3 and 3 %; EC3 = 1.51 %
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Table 1 below

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

446

14167

7674

2110

620

2

426

13137

6631

1899

746

3

323

11796

3766

1691

583

4

562

12366

5221

2541

706

5

480

15388

5863

2206

1027

 Group

mean

447

13371

5831

2089

736

SI

1.00

29.88

13.03

4.67

1.65

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

22.1

31.1

26.9

23.9

23.5

2

23.2

27.8

25.7

24.2

24.0

3

22.1

32.1

25.5

25.1

22.8

 4

24.8

29.5

26.4

25.8

22.7

5

22.5

38.1

26.4

24.1

21.6

 Group mean

22.94

31.72*

26.18*

24.62*

22.92

SD

1.13

3.92

0.57

0.80

0.91

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

447

1.0

22.94

PC

13371

29.88+

31.72*

30%

5831

13.03+

26.18*

3%

2089

4.67+

24.62

0.3%

736

1.65

22.92

Bold figures with + = values >= 3

NC = vehicle control; PC = positive control

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Pitch, Petroleum Tar, is 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 significant irritation. EC3 is calculated to be 1.51 %.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitising potential of pitch, petroleum, arom. was tested in a mouse local lymph node assay. Application of test substance to the ear of mice resulted in enhanced primary proliferation of lymphocytes in the lymph nodes draining the site of application measured by incorporation of radioactive labelling. Stimulation index (SI) was above 3 (4.61) at a test substance concentration of 3 %. An EC3 of 1.51 % was calculated by linear interpolation between the test substance concentration just below and above a SI of 3 (0.3 % and 3 %, respectively).


Migrated from Short description of key information:
Pitch, petroleum, arom. was demonstrated to exert a strong sensitising effect in a LLNA (local-lymph node assay). EC3 was 1.51 %.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:
Migrated from Short description of key information:
Experimental data on respiratory sensitisation could not be located. From occupational experience, there is no evidence of a sensitising potential to humans.

Justification for classification or non-classification

According to Regulation (EC) 1272/2008, classification is required, based on experimental evidence (EC3 of 1.51 % in a mouse LLNA): Skin Sens. 1A.