Ceraphyl 55

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

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

other: clear colourless liquid

Details on test material:

- Name of test material (as cited in study report): Ceraphyl 55
- Data of manufacture: 29 July 2003
- Physical state: Clear colourless liquid
- Analytical purity: 99.77%
- Impurities (identity and concentrations): 0.23% as isotridecanol
- Lot/batch No.: P1743
- Expiration date of the lot/batch: January 2006
- Storage condition of test material: Room temperature, in the dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0.1 mL volume additions were used for plate-incorporation treatment and 0.05 mL volume additions were used for pre-incubation treatments
Final concentration (µg/plate);
78.13
156.3
312.5
625
1250
2500
5000

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

yes

Remarks:

Ethanol

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

Toxicity Range-Finder Experiment
Triplicate plates with and without S9 mix were used. Negative (solvent) and positive controls were included in quintuplicate and triplicate, respectively. An initial Range Finder test was performed using TA100 strain only. Ceraphyl 55 was tested at 1.6, 40, 200, 100, 5000 µg/plate with and without metabolic activation (positive and negative controls included). The plating was achieved by adding 2.5 mL molten agar at 46±1C:
- 0.1 mL bacterial culture
- 0.1 mL test article solution
- 0.5 mL 10% S-9 mix or buffer solution
The culture were mixed and placed on agar plates. The plates were incubated at 37±1C in the dark for 3 days and then checked for revertant colonies.
Mutation Experiment
Two experiments were conducted using four strains of Salmonella Typhimurium and one strain Escherichia coli, with and without S9. The first experiment showed negative results; therefore the second experiment included a pre-incubation step. The test article (or control solution), bacteria and S9 mix were mixed together and incubated for 1 hour at 37±1C with shaking, before addition of 2.5 molten agar at 43±1C. Plating followed the normal procedure. In the second experiment, the volume addition in the pre-incubation treatments was reduced to 0.05 mL due to the solvent used (Ethanol).
Ethanol and other organic solvents are toxic at 0.1 mL, therefore 0.05 mL was chosen to minimise the possibility of toxicity.
Experiment 2 was repeated using WP2 uvrA strain with and without S9.

Evaluation criteria:

The assay was considered valid if:
1. The mean negative control counts fell within the normal ranges (Laboratory database)
2. The positive control chemicals induced clear increase in revertant numbers confirming discrimination between different strains, and an active S9 preparation
3. No more than 5% of the plates were lost due to contamination or other events.
4. A dose related increase in revertant numbers was observed that was at least two times the mean negative control counts for strain TA98, TA100 and WP2 uvrA and at least three times the mean negative control counts for strain TA1535 and TA1537.
5. The positive control has to be reproducible.

Statistics:

Mean and standard deviations were calculated from all individual plates of all experiments.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test article was observed following treatment of the two highest doses, both with and without S9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test article Ceraphyl 55 does not induce mutation in Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli ( WP2 uvrA).

Executive summary:

The objective of the study was to evaluate the mutagenic activity of the test material Ceraphyl 55 using four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli ( WP2 uvrA), both with and without S9. An initial range finder toxicity study was performed using only TA100 strain. No evidence of toxicity was observed at concentration of 1.6, 8, 40, 200, 1000 and 5000 µg/plate. Precipitation was observed at the two highest doses, with or without S9. The Experiment 1 included all strains and the same doses tested in the range finder test and as previously, no evidence of toxicity was observed at concentration of 1.6, 8, 40, 200, 1000 and 5000µ/plate. Precipitation of the test material was observed at the highest dose, with or without S9. The Experiment 2 was performed using all strains with and without S9 and a dose range from 78.13 to 5000 µg/plate. A pre-incubation step was included to increase the range of mutagenic chemicals able to induce positive responses in this test system. No evidence of toxicity was observed. Test material precipitation was observed at the two highest doses, both with or without S9. The negative and positive controls were included all gave results that fell within the acceptable range. The result of the test shows no increase in revertant numbers in any strain treatment with or without metabolic activation. In conclusion, Ceraphyl 55 is not mutagenic in Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli ( WP2 uvrA), both with and without S9.