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Toxicological information

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 26 May 2011 and 25 July 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study is conducted in compliance with agreed protocols, with no deviations from standard test guidelines and no methodological deficiencies. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. The reliability has been amended in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. Read-across is justified on the following basis: In accordance with Annex XI, Section 1.5 it is considered to be adequate and reliable to fulfil the information requirements for the endpoint ‘mutagenicity’ by applying a read-across approach from data generated on the substance iron orthophosphate (CAS # 10045-86-0). Both substances are relatively insoluble inorganic ferric (Fe3+) compounds. In conditions where the substances have limited solubility/bioavailability; ionisation to the Fe cation and the orthophosphate cation (iron orthophosphate) or pyrophosphate cation (tetrairon tris(pyrophosphate) will occur. In biological systems (i.e. in the presence of alkaline phosphatase) the pyrophosphate will be broken down into orthophosphate. It is considered that the Fe3+ cation is of most relevance when considering the genotoxic potential of the test material and as iron orthophosphate is slightly more soluble this substance is a good candidate for read-across. The study is considered to be adequate and reliable for the purpose of registration under REACH (Regulation (EC) No. 1907/2006) and for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 20 July 2010, Date of signature: 29 October 2010
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: 'solid'
Details on test material:
Sponsor's identification : IP 27 Iron orthophosphate
Description : Off white solid
Batch number : MV3395
Purity : 100%
Date received : 11 May 2011
Expiry date : 03 March 2014
Storage conditions : Room temperature, in the dark

Method

Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):

- Type and identity of media:
RPMI 1640

- Properly maintained:
yes

- Periodically checked for Mycoplasma contamination:
yes

- Periodically checked for karyotype stability:
no

- Periodically "cleansed" against high spontaneous background:
yes
:
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
the maximum dose level was (10mM) 1508 µg/ml
Vehicle and positive controls were used in parallel with the test item. Solvent (R0) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batch 0001423147 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation
Vehicle:

- Vehicle(s)/solvent(s) used:
Solvent (R0) treatment groups were used as the vehicle controls.


- Justification for choice of solvent/vehicle:
Formed the best doseable suspension
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
Solvent (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Negative controls:
yes
Solvent controls:
yes
Remarks:
Solvent (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation

Migrated to IUCLID6: :
Details on test system and conditions:
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese EPA/METI/MHLW guidelines for testing of new chemical substances.

Methods. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-Hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at six dose levels using a 4 Hour exposure group in the presence of metabolic activation (1% S9) and a 24 Hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test and for the first experiment was 23.56 to 1508 µg/ml in the absence and presence of metabolic activation. For the second experiment the dose range was 94.25 to1508 µg/ml in the absenceand presence of metabolic activation.

The maximum dose level used in the mutagenicity test was the maximum recommended dose (10mM) of 1508 µg/ml. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
Evaluation criteria:

Please see ''any other information on materials and methods incl. tables'' section.
Statistics:
Please see ''any other information on materials and methods incl. tables'' section.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: Thymidine Kinase TK+/-, locus of the L5178Y mouse lymphoma cell line
Remarks:
Migrated from field 'Test system'.
Additional information on results:
The dose range of the test item used in the Preliminary Toxicity Test was 5.89 to 1508 µg/ml
A precipitate was observed in all test item dose levels at the end of the exposure periods. Moderate reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item were observed in the 4-Hour exposure groups both in the absence and presence of metabolic activation when compared to the concurrent vehicle control groups. No marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item were observed in the 24-Hour exposure group when compared to the concurrent vehicle control group. Because of the reduction in %RSG observed in the 4-hour cultures with and without S9 the maximum dose level selected for the mutagenicity test was 1508 µg/ml, the 10 mM limit dose level
Mutagenicity Test

A summary of the results from the test is presented in attached in Table 1

Experiment 1
The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.
As was seen in the preliminary toxicity test a precipitate was observed in all test item dose levels and in both exposure groups at the end of the exposure period.
There was evidence of moderate test item-induced toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG values (Tables 3 and 6). There was no evidence of reductions in viability (%V) in both the absence and presence of metabolic activation, indicating that residual toxicity had not occurred (Tables 3 and 6). Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).
The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). A precipitate of test item was observed at and above 23.56 µg/ml.
The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.
Experiment 2
The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13.
As was seen in the preliminary toxicity test a precipitate was observed in all test item dose levels and in both exposure groups at the end of the exposure period.
As was seen previously, there was evidence of moderate toxicity in both the absence and presence of metabolic activation following exposure to the test item, as indicated by the %RSG values (Tables 9 and 12). There was no evidence of reductions in viability (%V) in both the absence and presence of metabolic activation, indicating that no residual toxicity had occurred (Tables 9 and 12). Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).
The 24-Hour exposure without metabolic activation demonstrated that the extended time point had no marked effect on the toxicity of the test item.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).
The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). All test item mutation values were within the acceptable range for vehicle control cultures. A precipitate of test item was observed at and above 94.25 µg/ml.
The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Any other information on results incl. tables

Please see Attached ''Tables 1 -10''

Due to the nature and quantitiy of tables it was not possible to insert them into this section.

Table 1 Summary of Results

Table 2 Cell and 96-Well Plate Counts: Experiment 1 (-S9) 4-Hour Exposure

Table 3 Statistical Analysis: Experiment 1 (-S9) 4-Hour Exposure

Table 4 Large and Small Colonies Analysis: Experiment 1 (-S9) 4-Hour Exposure

Table 5 Cell and 96-Well Plate Counts: Experiment 1 (+S9) 4-Hour Exposure

Table 6 Statistical Analysis: Experiment 1 (+S9) 4-Hour Exposure

Table 7 Large and Small Colonies Analysis: Experiment 1 (+S9) 4-Hour Exposure

Table 8 Cell and 96-Well Plate Counts: Experiment 2 (-S9) 24-Hour Exposure

Table 9 Statistical Analysis: Experiment 2 (-S9) 24-Hour Exposure

Table 10 Large and Small Colonies Analysis: Experiment 2 (-S9) 24-Hour Exposure

Table 11 Cell and 96-Well Plate Counts: Experiment 2 (+S9) 4-Hour Exposure

Table 12 Statistical Analysis: Experiment 2 (+S9) 4-Hour Exposure

Table 13 Large and Small Colonies Analysis: Experiment 2 (+S9) 4-Hour Exposure

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative Non-mutagenic

Conclusion. The test item did not induce any statistically significant or dose related increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test. This study is considered to be scientifically justified for use as a key study under Regulation (EC) No. 1907/2006 (REACH).