Tar, Pinus Sylvestris L. A complex combination distillate produced from trunks, stumps and roots of Pinus sylvestris L. by a traditional pine tar pit, kiln or retort process.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experiment was conducted between 14.3.-21.3.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Bacterial reverse mutation test using Salmonella typhimurium and Escherichia coli

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Tar, pine

EC Number:

232-374-8

EC Name:

Tar, pine

Cas Number:

8011-48-1

IUPAC Name:

Tar, pine

Test material form:

liquid

Method

Test concentrations with justification for top dose:

A stock solution for each assay was prepared at 100 mg/mL in DMSO. The highest dose tested was 5 000 μg / plate.
Doses of 5 000, 1 500, 500, 150 and 50 μg/plate were used from solutions of the test item Pine tar / Tar, Pinus sylvestris

Vehicle / solvent:

Vehicle used to solubilize test item ;DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Details on test system and experimental conditions:

Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1-9 x109 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar, maintained supercooled at 45° C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).
Escherichia coli strain : in a test tube 0.1 mL of the bacterial suspension containing 1-9 x 109 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled in 45° C containing 5% (v/v) of nutrient broth n° 2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL.
Plates are incubated at 37° C over a 48-72 hour period. The number of revertant colonies per plate is counted.

Evaluation criteria:

Ensure that the criteria of validity of the study are well respected namely :
-the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
-the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
-the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
-the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
-Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

Statistics:

After a 48-72 hour incubation period at 37° C, revertant colonies are counted in each plate.
Data are presented as the number of revertant colonies (mean  standard deviation) per plate.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: his

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Doses (5 000, 1 500, 500, 150 and 50 μg/plate) performed from solutions of the test item Pine tar / Tar, Pinus sylvestris, do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA-) (pKM 101) without, or with metabolic activation, according to the OECD Guidelines n°471.

Remarks on result:

other: no mutagenic change

Applicant's summary and conclusion

Conclusions:

Doses (5 000, 1 500, 500, 150 and 50 μg/plate) prepared from solutions of the test item Pine tar / Tar, Pinus sylvestris, do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101) without, or with metabolic activation, according to the OECD Guidelines n° 471.

Executive summary:

Test item, pine tar was tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out. For the two assays, negative and positive controls were carried out in parallel.

Doses (5 000, 1 500, 500, 150 and 50 μg/plate) prepared from solutions of the test item Pine tar / Tar, Pinus sylvestris, do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101) without, or with metabolic activation, according to the OECD Guidelines n° 471.