Tar, coal, high-temp.

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:

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)

Principles of method if other than guideline:

Determination of test concentration-related stimulation indices (SI) and the effective concentration (EC) inducing a stimulation index (SI) of 3.

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:

Tar, coal, high-temp.

EC Number:

266-024-0

EC Name:

Tar, coal, high-temp.

Cas Number:

65996-89-6

Molecular formula:

not applicable

IUPAC Name:

naphthalene

Details on test material:

- Name of test material (as cited in study report): Coal Tar
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Substance type: organic
- Physical state: liquid, viscous
- 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: 19.4 - 21.8 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: dispersion/emulsion- Irritation: yes
- Lymph node proliferation response: not examinedMAIN

STUDYANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response (on primary proliferation of lymphocytes by incorporation of 3H-methyl thymidine), Simulation index (SI) is ≥ 3, and response increases in dose-related mannerCriteria 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

Key result

Parameter:

EC3

Remarks:

[%]

Value:

< 0.3

Test group / Remarks:

estimated

Remarks on result:

other: concluded to be not irritating based on ear effect

Results 2

Key result

Parameter:

SI

Value:

7.36

Test group / Remarks:

0.3% test material in vehicle

Remarks on result:

other: not irritating based on ear effect

Results 3

Key result

Parameter:

SI

Value:

13.52

Test group / Remarks:

3% test material in vehicle

Results 4

Key result

Parameter:

SI

Value:

19.44

Test group / Remarks:

30% test material in vehicle

Results 5

Parameter:

SI

Value:

1

Test group / Remarks:

neg. control (vehicle)

Results 6

Parameter:

SI

Value:

16.94

Test group / Remarks:

pos. control

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA / SI DETERMINATION
see "Any other information on results"

EC3 CALCULATION
In this case, no reasonable calculation based on the algorithm below is possible, because there is no highest concentration that fails to produce an SI of 3: The SI for all test concentrations are significantly above 3. Hence the estimated EC is << 0.3%.

Base algorithm:
EC3 = c + [(3 -Sc)/(Sa-Sc)] * (a-c) with a = the lowest concentration giving stimulation SI >3; Sa = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; Sc = the actual stimulation index caused by c. (Note: The vehicle control data (at SI = 1) should not be used for the c and Sc.)
References:
Kimber, I.; Basketter, D.A.; Berthold, K.; Butler, M., et al. 2001:Skin sensitization testing in potency and risk assessment. Toxicol. Sciences, 59, 198 - 208 (p. 203)
Basketter et al. 1999:A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. Journal of Applied Toxicology, 19, 261–266

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	348	3414	4134	2755	1969
2	256	5547	6021	3491	1834
3	213	4808	5040	3536	1615
4	196	3278	4154	3467	2004
5	174	3045	3712	2793	1309
Group mean	237	4018	4613	3208	1746
SI	1.00	16.94	19.44	13.52	7.36

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	21.8	32.9	37.7	25.0	23.0
2	22.4	34.2	32.1	26.2	22.3
3	21.9	38.5	34.0	25.5	22.8
4	22.1	26.9	36.2	24.5	23.3
5	23.1	32.6	34.1	25.5	22.2
Group mean	22.26	33.02*	34.82*	25.34*	22.72
SD	0.52	4.16	2.17	0.63	0.47

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	238	1.0	22.26
PC	4018	16.94+	33.02
30%	4612	19.44+	34.82
3%	3208	13.52+	25.34
0.3%	1746	7.36+	22.72

Bold figures with + = values >= 3

NC = vehicle control; PC = positive control

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Coal tar considered to exhibit a sensitising potential, supported by the fact that a significant stimulatory effect was observed at a low dose that failed to produce visible irritation. The EC3 (Effective Concentration inducing a Stimulation Index SI of 3) is distinctly below 0.3%, hence significantly below the cut-off concentration of 2% for classification of category 1A skin sensitiser.