RAL 3.0

Genetic toxicity: in vitro

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Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Furthermore, a well-conducted (equivalent protocol compared to relevant OECD guideline) chromosome aberration study is available (Pharmakon Research International, 1991b).

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: : GLP compliant study performed to a method equivalent to the relevant guidelines, well conducted, no limitations in reporting and fully adequate for assessment

Cross-reference

Reason / purpose for cross-reference:

read-across source

Remarks:

The read-across can be performed because trisodium hexafluoroaluminate is component parts of RAL 3.0, and is the only component with possible negative effects for human health

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Remarks:

Furthermore, a well-conducted (equivalent protocol compared to relevant OECD guideline) chromosome aberration study is available (Pharmakon Research International, 1991b).

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other:

Remarks:

GLP compliant study performed to a method equivalent to the relevant guidelines, well conducted, no limitations in reporting and fully adequate for assessment

Reason / purpose for cross-reference:

read-across source

Remarks:

The read-across can be performed because trisodium hexafluoroaluminate is component parts of RAL 3.0, and is the only component with possible negative effects for human health.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Kryocide, Lot #86-12, was received by Pharmakon Research International, Inc. Fifty grams of the test article were submitted with the physical description of a white powder.
For the purposes of this study, the test article was stored at room temperature in the container received from the sponsor. All required dilutions
were prepared in water just prior to dosing. At the time of testing, Kryocide was described as a white powder, hence, there was no apparent change in its physical state during the storage.

Species / strain / cell type:

lymphocytes:

Remarks:

human

Metabolic activation:

with and without

Metabolic activation system:

rat S9-mix

Test concentrations with justification for top dose:

1, 5, 25, 50, 75, 100, 175, 250, 500 and 1000 µg/ml

Vehicle / solvent:

HPLC Grade Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C and cyclophosphamide

Details on test system and experimental conditions:

Kryocide was evaluated for cytotoxicity in human lymphocyte cultures utilizing cell proliferation kinetics and mitotic index (MI) as parameters. Whole human venous blood was drawn aseptically from a healthy donor. Kryocide was evaluated doses of 1, 5, 25, 50, 75, 100, 175, 250, 500 and 1000 in rat hepatocytes at dose levels up to 1000 µg/ml with and without an exogenous metabolic activation system (S9 mix). Concurrent untreated and solvent (HPLC Grade Water) controls were also evaluated. Based on the cytogenetic findings, the doses selected for the chromosome aberration assay were 100, 500 and 1000 µg/ml. Duplicate cultures of whole blood from the same donor were used for each test point.

Human lymphocytes were exposed to Kryocide at three different phases of the cell cycle (G0/G1, 2 and S) after lymphocytic culture initiation. Each set of cultures was harvested at the appropriate scheduled time. The slides were prepared for metaphase analysis according to standard methods. Fifty (50) metaphases were scored from each culture and data from duplicate cultures pooled for analysis. Treatment times were analyzed separately.

Evaluation criteria:

Assessment of a test article as positive is based upon its ability to produce a statistically significant increase in the frequency of chromosomal aberration in test cultures as compared to the solvent control and/or a dose response pattern

Statistics:

Chi-square analysis was done comparing each data point to its concurrent solvent control. Statistically significant differences in aberrations/cell were determined by pooling the data from the two cultures per data point and conductin g one-tailed t tests comparing treated cultures with their concurrent solvent controls.

Species / strain:

lymphocytes: human

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:

The results of the cytotoxicity evaluation indicated that Kryocide did not result in a significant increase in APT or a significant mitotic depression at any dose evaluated. Therefore, based on these results, 100, 500 and 1000 µg/ml were selected to be evaluated in the human lymphocyte assay with and without S9 mix at the G0/G1, G2 and S phases.

Treatment with Kryocide at 100, 500 and 1000 µg/ml with and without S-9 mix induced no statistically significant increases in aberrations/cell or proportion of aberrant metaphases at any dose level tested

Remarks on result:

other: all strains/cell types tested

Conclusions:

After performing chromosome aberration study in mammalian cells no adverse effect observed (negative).
RAL 3.0 is not harmful.

Executive summary:

After performing chromosome aberration study in mammalian cells no adverse effect observed (negative).

RAL 3.0 is not harmful.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Reference

Name:

Unnamed

Type:

impurity

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Cryolite
- Substance type: slighty coloured powder
- Analytical purity: 96.9%
- Composition of test material, percentage of components: Na 31%, Al 12.6%, F 53.3%
- Storage condition of test material: room temperature

Specific details on test material used for the study:

Kryocide, Lot #86-12, was received by Pharmakon Research International, Inc. Fifty grams of the test article were submitted with the physical description of a white powder.
For the purposes of this study, the test article was stored at room temperature in the container received from the sponsor. All required dilutions
were prepared in water just prior to dosing. At the time of testing, Kryocide was described as a white powder, hence, there was no apparent change in its physical state during the storage

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

rat S9-mix

Test concentrations with justification for top dose:

1, 5, 25, 50, 75, 100, 175, 250, 500 and 1000 µg/ml

Vehicle / solvent:

HPLC Grade Water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C and cyclophosphamide

Details on test system and experimental conditions:

Kryocide was evaluated for cytotoxicity in human lymphocyte cultures utilizing cell proliferation kinetics and mitotic index (MI) as parameters. Whole human venous blood was drawn aseptically from a healthy donor. Kryocide was evaluated doses of 1, 5, 25, 50, 75, 100, 175, 250, 500 and 1000 in rat hepatocytes at dose levels up to 1000 µg/ml with and without an exogenous metabolic activation system (S9 mix). Concurrent untreated and solvent (HPLC Grade Water) controls were also evaluated. Based on the cytogenetic findings, the doses selected for the chromosome aberration assay were 100, 500 and 1000 µg/ml. Duplicate cultures of whole blood from the same donor were used for each test point.

Human lymphocytes were exposed to Kryocide at three different phases of the cell cycle (G0/G1, 2 and S) after lymphocytic culture initiation. Each set of cultures was harvested at the appropriate scheduled time. The slides were prepared for metaphase analysis according to standard methods. Fifty (50) metaphases were scored from each culture and data from duplicate cultures pooled for analysis. Treatment times were analyzed separately.

Evaluation criteria:

Assessment of a test article as positive is based upon its ability to produce a statistically significant increase in the frequency of chromosomal aberration in test cultures as compared to the solvent control and/or a dose response pattern.

Statistics:

Chi-square analysis was done comparing each data point to its concurrent solvent control. Statistically significant differences in aberrations/cell were determined by pooling the data from the two cultures per data point and conductin g one-tailed t tests comparing treated cultures with their concurrent solvent controls.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

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:

The results of the cytotoxicity evaluation indicated that Kryocide did not result in a significant increase in APT or a significant mitotic depression at any dose evaluated. Therefore, based on these results, 100, 500 and 1000 µg/ml were selected to be evaluated in the human lymphocyte assay with and without S9 mix at the G0/G1, G2 and S phases.

Treatment with Kryocide at 100, 500 and 1000 µg/ml with and without S-9 mix induced no statistically significant increases in aberrations/cell or proportion of aberrant metaphases at any dose level tested.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

RAL 3.0 is not dangerous related to genetic toxicology.
No adverse effect observed (negative)

Executive summary:

After performing chromosome aberration study in mammalian cells no adverse effect observed (negative).

RAL 3.0 is not harmful.