Creosote oil, acenaphthene fraction

Endpoint summary

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: 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

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)].

Parameter:

SI

Remarks on result:

other: Concentration = 0.3% / 3% / 30% SI = 1.42 / 4.22 / 5.88 EC3 = 1.82 % Details see Table 2 below

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Concentration / 0.3% / 3% / 30% Group mean / 857 / 2538 / 3534 Details 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	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

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Wash oil considered to exhibit a clear sensitising potential in the LLNA, supported by the fact that a significant stimulatory effect was observed at a low dose (3 %) that failed to produce visible irritation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

High doses of wash oil (3 and 30 %) induced a weak but dose-related increase in lymphocyte proliferation in the LLNA assay (SI = 4.22 and 5.88, respectively) without evidence of an irritating potential. The finding is not considered to be a false positive result. Hence, wash oil should be regarded as a potential skin sensitiser: The EC3 is 1.82 % (calculated by interpolation), hence wash oil is to be classified as Skin Sens. 1A according to Regulation (EC) 1272/2008.

Migrated from Short description of key information:
In a mouse local lymph node assay, creosote oil, acenaphthene fraction (wash oil) showed a sensitising effect with an EC3 < 2.

Justification for classification or non-classification

Due to the positive result in the LLNA with EC3 of 1.82 % (i.e. below the cut-off limit of 2 %), wash oil has to be classified as skin sensitiser Cat. 1A (criteria of Regulation (EC) No 1272/2008).