Ceraphyl 55

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The three studies available on the test material Ceraphyl 55, two Ames/Salmonella tests and one CHO test show no mutagenic nor chromosome aberration activity.

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:

key study

Reliability:

1 (reliable without restriction)

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

Species / strain / cell type:

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

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

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.

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'.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Four in vitro genotoxicity studies are available on the test material Ceraphyl 55.

The test material Ceraphyl 55 was evaluated in the Ames/Salmonella Plate Incorporation Assay to determine its ability to induce reverse mutations in 5 strains of Salmonella Typhimurium (Pharmakon, 1987). A pre-test was conducted by treating duplicate cultures of strains TA1538 and TA100 at Ceraphyl 55 dose level of 50.0, 167, 500, 1670 ug/plate. None of the selected doses displayed toxicity. Inhibited growth was observed at dose level of 5000 ug/plate. At dose level of ≥ 500 the test article coalesced from solution upon addition to top agar. Based on these results, the test material was tested in triplicate cultures under the same pre-test condition at doses of 50.0, 167, 500, 1670, 3330 or 5000 ug/plate

, with and without S9. An extra dose was tested in the event of unacceptable high toxicity at the highest dose level. All positive and negative controls were within acceptable limits. The results of test showed that Ceraphyl 55 did not induce reverse mutations in Ames/Salmonella test, therefore it is considered to be not genotoxic.

The objective of the second 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 (Covance, 2004). An initial Range Finder Toxicity 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 of the test material was observed at the last two highest doses, with or without S9. The Experiment 1 included all strains and the same doses tested in the Range Finder test. As in the range-finder study, no evidence of toxicity was observed at concentrations of 1.6, 8, 40, 200, 1000 and 5000µg/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 µ/plate. A pre-incubation step was also included to increase the range of mutagenic chemicals able to induce positive response in this test system. No evidence of toxicity was observed. Test material precipitation was observed at the last two highest doses, with or without S9. The result for the negative and positive control materials 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 was not mutagenic test material in Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli ( WP2 uvrA), both with and without S9.

In addition, a test on Chinese hamster ovary (CHO) cells was conducted by Covance (2004) in order to investigate the potential to induce chromosome aberrations. Two experiments were performed. In the first experiment, cells were treated for three hours with and without metabolic activation (S-9) followed by a 17- hours recovery period to harvest (3 +17). The highest concentration chosen for analyses was 41.231µg/mL in the absence of S-9 and 600µg/mL in the presence of S-9. The highest concentration, 600.0 µg/mL was in excess of the solubility limit of the test article in culture medium. In the second experiment, cells were treated in the absence of S-9 continuously for 20 hours and in the presence of S-9 for 3 hours followed by 17 -hour recovery period prior to harvest (3 +17). The highest concentration chosen for analyses was 32.77µg/mL in the absence of S-9 and 600µg/mL in the presence of S-9. Solvent and positive controls were included in the test system in both experiments. The results of the two experiments showed no increases in the frequency of cells with numerical aberration, which exceed the historical negative control range, both with and without metabolic activation. In conclusion, Ceraphyl 55 did not induce chromosome aberration in cultured Chinese hamster ovary (CHO) cells when tested up to its limit of cytotoxicity or in excess of the solubility limit.

A fourth experiment was conducted by BioReliance Corporation (2013). Ceraphyl 55was investigatedfor its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of a metabolic activation system (S9).

A preliminary solubility testing was conducted using concentrations of 8.30, 16.6, 33.2, 66.4, 133, 266, 531, 1060, 2130 and 4250 µg/mL with and without S9 (the maximum concentration evaluated approximated the10 mM limit doseforthisassay). All test article concentrations, as well as the vehicle control, were evaluated in single cultures. The result showed thatCeraphyl 55 was freely soluble at all concentrations evaluated, and little or no cytotoxicity was observed. 

The concentrations used in the definitive mutagenicity assay were 531, 1060, 2130, 3190 and 4250 µg/mL with and without S9. All test substance concentrations, the positive and vehicle controls, were evaluated in duplicate cultures. Ceraphyl 55 was found to be incompletely soluble at the beginning of treatmentat a concentration of 4250 µg/mL, but became freely soluble by the end of treatment. Results showed no significant increases in mutant frequency, as compared to the concurrent vehicle controls at any concentration tested with or without S9 (p > 0.05). As expect, the positive controls induced significant increases in mutant frequency (p < 0.01).

An independent confirmatory assay was conducted at concentrations of 531, 1060, 2130, 3190 and 4250 µg/mL with and without S9. All test article concentrations, positive and vehicle controls, were tested in duplicate cultures. The results showed that Ceraphyl 55 was freely soluble at all concentrations evaluated, and little or no cytotoxicity was observed (average adjusted relative survivals at 4250 µg/mL were 102.0% and 74.6% with and without S9, respectively). No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration tested with or without S9 (p > 0.05; those cultures treated at a concentration of 2130 µg/mL with S9 were excluded from statistical evaluation due to loss of one of the replicate cultures). The positive controls induced significant increases in mutant frequency (p < 0.01).

All positive and vehicle control values were within acceptable ranges. All criteria for a valid assay were met. In conclusion, the test materialCeraphyl 55 resulted negative in theIn Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures under the condition of this study.

Justification for classification or non-classification

Ceraphyl 55 are not considered mutagenic and do not meet the criteria for classification and labelling under CLP EU Regulation 1272/2008.