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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation study in bacteria:

Key study: An in-vitro bacterial reverse assay (Ames test) was performed on OS2600 in accordance with OECD Guideline 471 (GLP study). Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100 and Escherichia coli, strain WP2 uvrA (pKM101), were exposed to OS2600 diluted in dimethyl sulphoxide (DMSO) up to 5000 µg/plate with and without metabolic activation. OS2600 did not induce a significant dose-related increase in the number of revertant colonies in any tested strain and therefore, it was determined to be non-mutagenic under the test conditions.

In-vitro gene mutation study in mammalian cells:

Key study: Read-across approach from experimental data on the analogue substance OS1600: An in-vitro mutation test using Mouse Lymphoma L5178Y Cells was performed in accordance with OECD Guideline 476 (GLP study). Based on the read-across approach from experimental data on the analogue substance OS1600 (where cultures were exposed up to 3435.5 µg/mL for 3 h and/or 24 h with and without metabolic activation and no increases in the mean mutant frequencies exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor), the OS2600 was determined to have no mutagenic potential under the test conditions.

In-vitro cytogenicity study in mammalian cells:

Key study. Read-across approach from experimental data on the analogue substance OS1600: A study was performed to assess the ability to induce chromosomal aberrations in human lymphocytes cultured in vitro in accordance with OECD Guideline 473 (GLP study). Based on the read-across approach from experimental results on the analogue OS1600 (where cultures were exposed >= 3250 µg/mL with metabolic activation for 3h caused an increase in the frequency of structural chromosome aberration, but instead, the results were negative for cultures treated without metabolic activation), it was concluded that the substance OS2600 could cause structural chromosome aberrations in the presence of metabolic activation under test the in-vitro cytogenetic test conditions.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-Mar-2012 to 15-Mar-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Of
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 1, 3, 10, 33, 66 and 100 µg/plate
With S9-mix: 1, 3, 10, 33, 100 and 200 µg/plate.
Experiment 2:
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate in DMSO for TA100
Positive control substance:
2-nitrofluorene
Remarks:
without S9, 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
sodium azide
Remarks:
without S9 : 5 µg/plate in saline for TA1535
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Key result
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate
Conclusions:
OS 2600 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
Executive summary:

OS 2600 was tested up to 5000 μg/plate in the Salmonella typhimurium reverse mutation assay in accordance with OECD Guideline 471 with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of metabolic activation. OS 2600 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from an analogue substance for which a guideline study (KIimish = 1) is available.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See reporting format for the analogue approach attached.
Reason / purpose:
read-across source
Key result
Species / strain:
lymphocytes: Human donor
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: Human donor
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Read-across from an analogue substance.
Remarks:
See summary and cross reference to source.

The data matrix is included in the reporting format attached.

Conclusions:
Based on the read-across approach from the analogue substance OS1600, it was concluded that the substance OS2600 could cause structural chromosome aberrations in the presence of metabolic activation.
Executive summary:

An in-vitro chromosome aberration test was performed with the analogue substance OS1600 in accordance with OECD Guideline 473. The analogue OS1600 showed evidence of causing an increase in the frequency of structural chromosome aberrations in the presence of S9 mix only in this in vitro cytogenetic test system under the experimental conditions described. Based on these results, the read-across approach was applied and it was concluded that the substance OS2600 could cause structural chromosome aberrations in the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from an analogue substance for which a guideline study (KIimish = 1) is available.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See reporting format for the analogue approach attached.
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Read across from an analogue substance.
Remarks:
See summary and cross reference to source.

The data matrix is included in the reporting format attached.

Conclusions:
Based on the read-across approach from the analogue substance OS1600, OS2600 was determined to be non-mutagenic in the in vitro cell mutation assay, under the experimental conditions described.
Executive summary:

An in-vitro mutation test using Mouse Lymphoma L5178Y Cells was performed with the analogue substance OS1600 in accordance with OECD Guideline 476. OS1600 did not demonstrate mutagenic potential in this in vitro cell mutation assay. Based on these results, the read-across approach was applied and the substance OS2600 was determined to be non-mutagenic in the in vitro cell mutation assay, under the experimental conditions described.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In-vivo micronucleus assay:

Key study: Read-across approach from experimental data on the analogue substance OS1600: An in-vivo micronucleus test in Crl: CD(SD) rats was performed according to OECD Guideline 474 (GLP study). Based on the read-across approach from the experimental data on the analogue substance OS1600 (no evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male rats when administered up to 500 mg/kg bw orally by gavage), the substance OS2600 was also determined to be negative for the in-vivo micronucleus test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from an analogue substance for which a guideline study (KIimish = 1) is available.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See attached reporting format.
Reason / purpose:
read-across source
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Read across from an analogue substance.
Remarks:
See summary and cross reference to source.

The data matrix is included in the reporting format attached.

Conclusions:
Based on the read-across approach from the analogue substance OS1600, OS2600 was determined not to cause an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD(SD) rats when administered orally by gavage up to an estimated dose of 2340 mg/kg bw.
Executive summary:

An in-vivo micronucleus test in Crl: CD(SD) rats was performed with the analogue substance OS1600 according to OECD Guideline 474. OS1600 did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes (MPCE) or bone marrow cell toxicity in male rats when administered orally by gavage in this in vivo test procedure. Based on these results, the read-across approach was applied and the substance OS2600 was also determined to be negative in the in-vivo micronucleus test under test conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Based on this battery of in vitro and in vivo test, the weight of evidence indicate that OS2600 is not genotoxic.

Additional information

In vivo and in vitro genetic toxicology studies indicate that OS2600 is not genotoxic.

Justification for classification or non-classification

Based on the available experimental data, OS2600 was determined to be non-genotoxic, and therefore it is not classified in accordance with CLP Regulation (EC) no. 1727/2008.