Pitch, petroleum, arom.

Skin sensitisation

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Pitch, petroleum, arom.

EC Number:

269-110-6

EC Name:

Pitch, petroleum, arom.

Cas Number:

68187-58-6

Molecular formula:

not applicable

IUPAC Name:

Pitch, petroleum, arom.

Details on test material:

- Name of test material (as cited in study report): pitch (petroleum tar)
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Substance type: organic
- Physical state: solid
- Stability under test conditions: WAF (water accommodated fraction) limited
- 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: ~ 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

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: 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)].

Results and discussion

In vivo (LLNA)

Results

Results 1

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 %

Results 2

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	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

Applicant's summary and conclusion

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 %.