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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Ames test (OECD 471): non-mutagenic with or without metabolic activation. Although this single data does not support the classification for mutagenicity, this substance needs to be classified for Muta 1B according to the most critical constituent in the substance (benzene). This classification conclusion is based on the harmonised classification entry of benzene and the maximum concentration of benzene 1.0 % (w/w) in the substance. Benzene has harmonized classification as Muta. 1B. Based on the CLP mixtures rules the general concentration limit (GCL) c ≥ 0.1 % triggers this substance to be classified as mutagenic Cat 1B.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
March 14, 2012 - May 03, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was regarded reliable without restriction since the study was conducted according to the OECD guideline 471 and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
A preliminary range-finding assay
TA100 and WP2uvr with and without metabolic activation (micrograms/plate)
5000, 1500, 500, 150, 50, 0

Main assay 1 (micrograms/plate)
Salmonella strain TA100 (without S9-mix) : 0.15, 0.5, 1.5, 5, 15, 50, 150
Salmonella strains TA98, TA1535, TA1537 (without S9-mix) : 0.5, 1.5, 5, 15, 50, 150, 500
All Salmonella strains (with S9-mix) and WP2uvrA (without S9-mix) : 1.5, 5, 15, 50, 150, 500, 1500
WP2uvrA (with S9-mix) : 50, 150, 500, 1500, 5000

Main assay 2 (micrograms/plate)
Salmonella strains TA100, TA1537 and TA98 (without S9-mix) : 0.15, 0.5, 1.5, 5, 15, 50
Salmonella strain TA1535 (without S9-mix) : 0.05, 0.15, 0.5, 1.5, 5, 15, 50
Salmonella strains TA100 and TA1537 (with S9-mix) : 1.5, 5, 15, 50, 150, 500
Salmonella strains TA1535 and TA98 (with S9-mix) : 5, 15, 50, 150, 500, 1500
WP2uvrA (without S9-mix) : 5, 15, 50, 150, 500, 1500, 5000
WP2uvrA (with S9-mix) : 50, 150, 500, 1500, 5000

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at 100 mg/ml.
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
Bacterial cultures and S-9 mix or phosphate buffer and vehicle or test item were incubated for 20 min at 37 deg. C. Two milliliters molten Top Agar, trace histidine or trypthophan, were added to the mixtures. The mixture was plated on Minimal Agar plates and incubated for 48 hours at 37 deg. c.

Evaluation criteria:
Following criteria are used for determining a positive result:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data as determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear negative or positive results, in some instances the data generated will prohibit making a definitive judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical analysis of data is conducted as determined by UKEMS (Mahon et al., 1989, UKEMS sub-committee on guidelines for mutagenicity testing, (Kirkland D J Ed.) Cambridge University Press Report, 26-65.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at test item concentrations 15 micrograms/plate or higher without metabolic activation and 150 micrograms/plate or higher with metabolic activation.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at test item concentrations 50 micrograms/plate or higher without metabolic activation and 500 micrograms/plate or higher with metabolic activation.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at test item concentrations 150 micrograms/plate or higher without metabolic activation. No cytotoxicity was observed with metabolic activation up to the test item concentration of 5000 micrograms per plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at test item concentrations 50 micrograms/plate or higher without metabolic activation and 500 micrograms/plate or higher with metabolic activation.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at test item concentrations 50 micrograms/plate or higher without metabolic activation and 150 micrograms/plate or higher with metabolic activation.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: immiscible
- Precipitation: no test item precipitation was observed on the plates at any of the doses tested


RANGE-FINDING/SCREENING STUDIES: The test item was toxic to TA100 from 50 and 500 micrograms per plate in the absence and presence of S9-mix respectively. No toxicity was observed to WP2uvrA.

COMPARISON WITH HISTORICAL CONTROL DATA: See attached background material.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test item was tested for mutagenic potential using in vitro bacterial reverse mutation test. The test substance did not exert mutagenic activity both in the presence and the absence of metabolic activation.
Executive summary:

The test item was tested for the mutagenic potential using in vitro bacterial reverse mutation test (Ames test)(corresponding to the OECD No. 471). A preliminary range-finding assay was performed using four strains of Salmonella typhimurium (TA100, TA1535, TA98 and TA1537) and one strain of E. Coli WPuvrA) up to a maximum dose of 5.0 mg/plate to determine the optimal non-toxic test dose. The test item was either tested up to the maximum recommended dose level of 5.0 mg/plate or the toxic limit depending o bacterial strain type and presence or absence of S9-mix.

Test item did not produce any significant increase of mutation frequency in any of these strains, with any dose of the test item, either in the absence or presence of metabolic activation. Based on the study results the test substance is considered to be non-mutagenic.

The test result is used as a key value in the hazard assessment.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

Weight of evidence approach was used to fulfil the guideline requirement for this endpoint. Assessment of genetic toxicity is based on thein vitrobacterial mutagenicity study conducted for the substance, the harmonized classification entry of the most critical constituent in the substance (benzene).

Ames study by Thompson, P. W. (2012) is considered reliable without restrictions as the study was performed in compliance with GLP and according to OECD guideline 471. This study was conducted by using four strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537) and one strain of E. coli (WP2) with and without metabolic activation. The substance was tested for its ability to induce mutations at the concentration of 5, 15, 50, 150, 500, 1500, 5000 micrograms/plate. Test item did not produce any significant increase in mutation frequency in any of these strains, with any dose of the test item, either in the absence or presence of metabolic activation. This study result would suggest that renewable hydrocarbons of wood origin (naphtha type fraction) is non-mutagenic.

 

The result of the Ames study was considered as false negative due to the testing difficulties, such as complex nature and volatilisation of the substance. Therefore, the Ames study result does not support the classification of this substance as a potential mutagenic. However, the classification is warranted based on the benzene concentration is the substance. The benzene concentration varies between 0.1 - 1.0 % (w/w). Benzene has harmonized classification as Muta. 1B. Based on the CLP mixtures rules the general concentration limit (GCL) c ≥ 0.1 % triggers this substance to be classified as mutagenic Cat 1B.


Justification for selection of genetic toxicity endpoint
The study conducted for the substance. Although this single data does not show any evidence of mutagenicity, this substance is classified for genotoxicity based on the most hazardous and critical constituent in the substance (benzene).

Justification for classification or non-classification

The renewable hydrocarbons of wood origin (naphtha type fraction) will be classified for Muta 1B H340 in accordance with the criteria of CLP Regulation 1272/2008 and for Muta. Cat. 2, R46 according to the EU directive 67/548/EEC.