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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 April, 2014 - 02 June, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(2008)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Strontium hydrogen phosphate
EC Number:
236-615-8
EC Name:
Strontium hydrogen phosphate
Cas Number:
13450-99-2
Molecular formula:
SrHPO4
IUPAC Name:
strontium hydrogen phosphate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): SrHPO4
- Description: White powder

Method

Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 17, 52, 164, 512 and 1600 µg/mL
Without S9-mix, 24 hours treatment: 17, 52, 164, 512 and 1600 µg/ml
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.05, 0.17, 0.5, 1.7, 5.2, 17, 52 and 164 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 0.05, 0.17, 0.5, 1.7, 5.2, 17, 52 and 164 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: At concentrations of 1.7 mg/ml and higher SrHPO4 was suspended in DMSO. At concentrations of 0.52 mg/ml and lower the test substance was dissolved in DMSO. Except in the second experiment where the test substance was still in suspension at 0.52 mg/ml. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. DMSO has been accepted and approved by authorities and international guidelines.

Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9. Treatment: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9. 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.


DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No data
- Effects of osmolality: No data
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 52 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to the precipitating dose levels of 52 µg/mL and above (up to 1600 µg/mL in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
Mutation frequencies of one of the solvent control cultures in the first experiment in the presence of S9-mix and one of the solvent control cultures in the second experiment were recorded to be outside the range of ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
Evaluation: The values of 48 and 44 per 10^6 survivors were below the lower limit of the range (50 per 10^6 survivors). The mutation frequencies were just below the lower limit of the range and clear negative results are observed in all experiments, therefore this deviation in the mutation frequency had no effect on the results of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to and including the highest tested precipitated dose levels in both experiments in the absence and presence of S9-mix.

POSITIVE CONTROLS
Mutation frequencies in cultures treated with positive control chemicals were increased by 17- and 13-fold for MMS in the absence of S9-mix, and 54-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay
Remarks on result:
other: strain/cell type: Test system L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative. It is concluded that SrHPO4 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the absence and presence of S9 -mix.
Executive summary:

The mouse lymphoma assay with SrHPO4 was conducted according to OECD 476 guideline and GLP principles.

No toxicity was observed up to and including the highest tested precipitated dose levels in both experiments in the absence and presence of S9-mix. In the absence of S9-mix, SrHPO4 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, SrHPO4 did not induce a significant increase in the mutation frequency in one experiment. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. It is concluded that SrHPO4 is not mutagenic in the TK mutation test system under the experimental conditions described in the absence and presence of S9 -mix.

 

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