Hydrocarbons, terpene processing by-products

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July - 6 August 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in accordance with OECD Guideline 471 with deviations: the temperature of the incubator rose to 38.1 °C overnight in Experiment 1; storage temperature of the test article was exceeded to 11.4 °C

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

the temperature of the incubator rose to 38.1 °C overnight in Experiment 1; storage temperature of the test article was exceeded to 11.4 °C

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hydrocarbons, terpene processing by-products

EC Number:

273-309-3

EC Name:

Hydrocarbons, terpene processing by-products

Cas Number:

68956-56-9

Molecular formula:

Not applicable for UVCB substance

IUPAC Name:

Hydrocarbons, terpene processing by-products

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Hydrocarbons, terpene processing by-products
- Physical state: Colourless liquid
- Analytical purity: 100 % (sum of terpene hydrocarbons: 89.1%)
- Lot/batch No.: I23-090312
- Expiration date: 9 March 2013
- Storage condition of test material: 2-8 °C, under nitrogen, protected from light

Method

Target gene:

His+ for S. typhimurium

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all strains (plate incorporation method)
Experiment 2: 0.4096, 1.024, 2.56, 6.4, 16, 40 and 100 µg/plate, with S9 mix (pre-incubation method) and without S9 mix (plate incorporation method) in all strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Dimethyl sulphoxide)
- Test article stock solutions were prepared by formulating test item in DMSO under subdued lighting conditions with the aid of vortex mixing, immediately prior to assay to give the maximum required treatment solution concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 5 h of initial formulation.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Nitrofluorene: 5 μg/plate for TA98; Sodium azide: 2 μg/plate for TA100 and TA1535; 9-Aminoacridine: 50 μg/plate for TA1537; mitomycin C: 0.2 μg/plate for TA102

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Benzo[a]pyrene: 10 μg/plate for TA98; 2-Aminoanthracene: 5 μg/plate for TA100, TA1535 and TA1537 and 20 μg/plate for TA102

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA.

METHOD OF APPLICATION: In agar (direct plate incorporation and preincubation method)

DURATION
- Preincubation period: 20 minutes at 37±1 °C
- Incubation period: 3 days at 37±1 °C for both direct plate incorporation and preincubation methods

NUMBER OF REPLICATIONS:
-3 plates/dose for treatment and positive controls
- 5 plates/dose for vehicle control

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn of treated plates was inspected for signs of toxicity.

OTHER: Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments).

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p ≤ 0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.
- The test article was considered positive in this assay if all of the above criteria were met.
- The test article was considered negative in this assay if none of the above criteria were met.
- Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

- Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None

CYTOTOXICITY:
- Experiment 1: Toxicity was observed at 50 μg/plate and above in all strains in the absence and presence of S-9.
- Experiment 2: Toxicity was observed at 16 μg/plate and above in strains TA100 and TA1535 in the presence of S-9; 40 μg/plate and above in strains TA100, TA1535 and TA102 in the absence of S-9 and strain TA1537 in the absence and presence of S-9; and at 100 μg/plate in strain TA98 in the absence and presence of S-9 and strain TA102 in the presence of S-9.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical data from the period February 2008 - July 2009.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

See attached Document for Tables of Results

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, Hydrocarbons, terpene processing by-products batch I23-090312 is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA102) strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) and were exposed to Hydrocarbons, terpene processing by-products batch I23-090312 at the following concentrations:

- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all strains (plate incorporation method)

- Experiment 2: 0.4096, 1.024, 2.56, 6.4, 16, 40 and 100 µg/plate, with S9 mix (pre-incubation method) and without S9 mix (plate incorporation method) in all strains

 

Metabolic activation system used in this test was 10% S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254. Vehicle and positive control groups were also included in mutagenicity tests.

In Experiment 1, toxicity was observed at 50 μg/plate and above in all strains in the absence and presence of S-9. In Experiment 2, toxicity was observed at 16 μg/plate and above in strains TA100 and TA1535 in the presence of S-9; 40 μg/plate and above in strains TA100, TA1535 and TA102 in the absence of S-9 and strain TA1537 in the absence and presence of S-9; and at 100 μg/plate in strain TA98 in the absence and presence of S-9 and strain TA102 in the presence of S-9. Following treatments of all the test strains in the absence and presence of S-9, only Experiment 2 treatments of strain TA102 in the presence of S-9 at 2.56 μg/plate resulted in an increase in revertant numbers that was statistically significant when the data were analysed at the 1 % level using Dunnett’s test, however this was not concentration related or reproducible and was of insufficient magnitude to be considered as clear evidence of mutagenic activity in this assay system. No other increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

 

Under the test conditions, Hydrocarbons, terpene processing by-products batch I23-090312 was not considered as mutagenic in this bacterial system.