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Administrative data

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 OS1600 in accordance with OECD Guideline 471. Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100 and Escherichia coli, strain WP2 uvrA (pKM101), were exposed to OS1600 diluted in dimethyl sulphoxide (DMSO) up to 5000 µg/plate. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats. It was concluded that OS1600 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

In-vitro gene mutation study in mammalian cells:

Key study: An in-vitro mutation test using Mouse Lymphoma L5178Y Cells was performed with OS1600 in accordance with OECD Guideline 476. Based on a preliminary test, cultures were exposed up to 3435.5 µg/mL for 3 h with and without metabolic activation and for 24 h without metabolic activation. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. It was concluded that OS 1600 did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

In-vitro cytogenicity study in mammalian cells:

Key study: A study was performed to assess the ability of OS 1600 to induce chromosomal aberrations in human lymphocytes cultured in vitro in accordance with OECD Guideline 473. Cultures were exposed up to 3000 µg/mL in the absence of S9 mix and up to 3435.5 µg/mL in the presence of S9 mix, for 3 h treatment period with 28 h recovery. It is concluded that OS 1600 has shown evidence of causing an increase in the frequency of structural chromosome aberrations at >= 3250 µg/mL, in the presence of S9 mix only, in this in vitro cytogenetic test system, under the experimental conditions described.

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
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 pKM 101
Metabolic activation:
with and without
Metabolic activation system:
liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Test 1: 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Test 2: 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- DMSO (test substance not stable in water)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: BaP, AAN, 2NF, NaN3, AAC, NQO as appropriate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) - 1st test; preincubation- 2nd test

DURATION
TEST 1: In agar (plate incorporation)
- Exposure duration: 72 hours at 37 ºC in petri dishes
TEST 2: Pre-incubation
- Preincubation period: 30 minutes at 37 ºC before the addition of the agar overlay.

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range; the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. To be considered positive, the test substance mustinduce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls
Statistics:
Not needed
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
All controls were within 99% of the historical range. All positive controls responded with a positive response.
Conclusions:
OS1600 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

An in-vitro bacterial reverse assay (Ames test) was performed on OS1600 in accordance with OECD Guideline 471. Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100 and Escherichia coli, strain WP2 uvrA (pKM101), were exposed to OS1600 diluted in dimethyl sulphoxide (DMSO) up to 5000 µg/plate. DMSO was also used as a negative control. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. No signs of toxicity were observed towards the tester strains in either mutation test following exposure to OS1600. No evidence of mutagenic activity was seen at any concentration of OS1600 in either mutation test. It was concluded that OS1600 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human blood from male donors
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human blood from two healthy non-smoking donors (pooled)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 supplemented with 10 % foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 µg/mL streptomycin and 2.0 mM glutamine. The cell supensions were incubated in a 5 % CO2 atmosphere.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Male rat livers dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver.
Test concentrations with justification for top dose:
In the presence and absence of S9 mix, 3 hour treatment, 18 hour recovery: 250, 500, 1000, 2000, 2250, 2500, 2750, 3000, 3250 and 3435.5 µg/mL.
Vehicle / solvent:
Dimethyl sulphoxide
Positive controls:
yes
Remarks:
In the absence of S-9
Positive control substance:
mitomycin C
Remarks:
0.2 ug/mL (3 hr. treatment)
Positive controls:
yes
Remarks:
In the presence of S-9
Positive control substance:
cyclophosphamide
Remarks:
5, 7.5, and 10 ug/mL (3 hr. treatment)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Remarks:
0.2 ug/mL
Details on test system and experimental conditions:
Vehicle, positive and treatment cultures were established approximately 48 hours after commencement of incubation of lymphocyte cultures. Duplicate cultures were prepared throughout for each 3 hour treatment in the absence and presence of S9 mix. All cultures were centrifuged and resuspended in fresh medium just before treatment.

In the absence of S9 mix, 50 µL aliquots of OS 1600 were added to give final concentrations of 34.62, 57.70, 96.17, 160.29, 267.14, 445.24, 742.07, 1236.78, 2061.3, 3435.5 µg/mL. DMSO was used as the vehicle control and Mitomycin C at a final concentration of 0.2 µg/mL was the positive control.

For treatments in the presence of S9 mix, 1 mL of medium was removed from each culture and discarded. This was replaced with 1 mL of S9 mix (2% v/v final concentration), followed by 50 µL of each test substance dilution (giving the same series of final concentrations as above). DMSO was used as the vehicle control and Cyclophosphamide at a range of final concentrations (5, 7.5 and 10) µg/mL was the positive control.

In both the absence and presence of S9 mix, no notable culture medium changes were observed after 3 hour treatment, when compared with the vehicle control. Following 3 hour treatment, cultures were centrifuged at 500g for 5 minutes. The supernatant removed and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours.

As an appropriate toxicity profile was not achieved, additional tests were conducted to achieve an acceptable concentration range in the absence and presence of S9 mix.

Concentrations of OS 1600 were as follows:
In the absence of S9 mix: 250, 500, 1000, 2000, 2250, 2500, 2750, 3000, 3250 and 3435.5 µg/mL.
In the presence of S9 mix: 250, 500, 1000, 2000, 2250, 2500, 2750, 3000, 3250 and 3435.5 µg/mL.

In both the absence and presence of S9 mix, no notable culture medium changes were observed after 3 hour treatment.

Two hours before the cells were harvested, mitotic activity was arrested by addition of (50 µL) Colcemid® to each culture at a final concentration of 0.1 µg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The cell pellets were treated with a hypotonic solution (0.075M KCl), prewarmed at 37°C. After a 10 minute period of incubation at 37°C, the suspensions were
centrifuged at 500g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear.

The pellets were resuspended, then centrifuged at 500g for 5 minutes and finally resuspended in a small (approximately 1 mL) volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the
cell pellets in fixative were stored at approximately 4°C until slide analysis was completed.
Evaluation criteria:
The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures, or cultures where there were no signs of cytotoxicity. From these results the concentration causing a decrease in mitotic index of at least 50% (where possible) of the vehicle control value was the highest concentration selected for metaphase analysis. Intermediate and low concentrations were also selected.

The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x1000 using an oil immersion objective. One hundred metaphase figures were examined from each culture. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen.
The vernier readings of all aberrant metaphase figures were recorded.
Statistics:
The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

D20s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002).

The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.0) which was developed in SAS (SAS INSTITUTE 2002).
Key result
Species / strain:
lymphocytes: Healthy donor
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: Healthy donor
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the absence of S9 mix following 3 hour treatment, OS 1600 caused a reduction in the mitotic index to 50% of the vehicle control value at 3000 µg/mL (determined by mitotic index). The concentrations selected for metaphase analysis were 500, 2250 and 3000 µg/mL. In the presence of S9 mix following 3 hour treatment, OS 1600 caused a reduction in the mitotic index to 76% of the vehicle control at 3435.5 µg/mL, the highest tested concentration. The concentrations selected for the metaphase analysis were 250, 3250 and 3435.5 µg/mL.

In the absence of S9 mix, OS 1600 caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the vehicle control.

All mean values for the vehicle control (DMSO) and OS 1600 treatment concentrations (with the exception of 3000 µg/mL, which lies outside the historical range), were within the laboratory historical control range, when taken at the 99% confidence limit.

In the presence of S9 mix, OS 1600 caused statistically significant increases at concentrations of 3250 µg/mL (p<0.01: including gaps) and 3435.5 µg/mL (p<0.001: including and p<0.01: excluding gaps) in the proportion of metaphase figures containing chromosomal aberrations, when compared with the vehicle control. No statistically significant increases were observed at the lowest tested treatment concentration of 250 µg/mL, when compared with the vehicle control.

All mean values for the vehicle control (DMSO), and the lowest tested OS 1600 treatment concentration of 250 µg/mL were within laboratory historical control range, when taken at the 99% confidence limit. However, at the higher tested treatment concentrations of 3250 µg/mL (p<0.01: including gaps) and 3435.5 µg/mL (p<0.001: including and p<0.01 excluding gaps) where statistically significant increases were observed, the mean values exceeded the laboratory historical control range, when taken at the 99% confidence limit. In addition, at 3435.5 µg/mL (10 mM), where relatively no cytotoxicity was observed (reduction in mitotic index of 76%, when compared with the vehicle control), a clear increase in major structural damage was observed.

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

No statistically significant increases in polyploid metaphases were observed during metaphase analysis.
Conclusions:
OS 1600 has shown 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.
Executive summary:

A study was performed to assess the ability of OS 1600 to induce chromosomal aberrations in human lymphocytes cultured in vitro in accordance with OECD Guideline 473. Cultures were exposed up to 3000 µg/mL in the absence of S9 mix and up to 3435.5 µg/mL in the presence of S9 mix, for 3 h treatment period with 28 h recovery. Two hours before the end of the incubation period, cell division was arrested using Colcemid, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. In the absence of S9 mix, OS 1600 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the vehicle control. In the presence of S9 mix, OS 1600 caused statistically significant increases at 3250 μg/mL and 3435.5 μg/mL in the proportion of metaphase figures containing chromosomal aberrations, when compared with the vehicle control. No statistically significant increases in the proportion of polyploid cells were observed. All positive control demonstrated the sensitivity of the test system and the efficacy of the S9 mix. It is concluded that OS 1600 has shown 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - 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
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
liver preparations (S9 mix) from male rats dosed with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Up to 3435.5 µg/mL
Vehicle / solvent:
dimethyl sulphoxide (DMSO)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Aliquots of 100 μL of test substance dilution, vehicle or positive control were added, then all cultures were incubated for 3 hours at 37ºC. Following the 3 h and 24 h exposure, the cells were washed once, resuspended in R10p to nominally 2 x 10+05 cells/mL and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted 2 x 10+05 cell/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 10+05 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 10+03 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air. After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates, the number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF).

DURATION
- Exposure duration: 3 h and 24 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): up to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS: Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants.
Evaluation criteria:
Tests were accepted on the basis of the following criteria:
Acceptance criteria for test substance:
The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (i.e. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.
Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 50 to 170 x 10-6.
The mean cloning efficiency was between 65 to 120%.
The mean suspension growth was between 8 to 32 on Day 2 following 3 hour treatments and between 32 to 180 on Day 2 following a 24 hour treatment.
Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.
Acceptance criteria for positive controls:
Positive controls showed an absolute increase in mean total MF above the mean concurrent vehicle control MF of at least 300 x 10-6. At least 40% of this was due to the number of small mutant colonies.
Mean RTG’s for the positive controls were greater than 10%.
There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity.
There was not excessive heterogeneity between replicate cultures.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p<0.001. Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend.
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
Cells were exposed to the test substance for 3 h with and without S9 mix and for 24 h without S9 mix. One culture was prepared for each concentration of the test substance for each test condition. Vehicle controls were tested in duplicate for each test condition. Aliquots of 50 μL of test substance dilution or vehicle were added to each culture prior to incubation for 3 h (continuous shaking at 37°C) or 24 h (static incubator, at 37°C, 5% (v/v) CO2). At the end of the 3 or 24 h exposure period, the cells were washed once, resuspended in R10p to nominally 2 x 10E+05 cells/mL (assuming no cell loss), incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E+05 cells/mL with R10p where necessary. There was evidence of toxicity in the preliminary toxicity test, so the maximum concentration tested in the 3 h exposure in both the absence and presence of S9 mix was 3435.5 μg/mL, and in the 24 hour exposure in the absence of S9 mix was 2000 μg/mL.

Following a 3 h exposure up to 3435.5 μg/mL test item, relative suspension growth (RSG) was reduced from 142 to 30% and from 123 to 59% in the absence and presence of S9 mix respectively. Following a 24 h exposure in the absence of S9 mix RSG was reduced from 131 to 1%. Following 3 h treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3 hour treatment in both the absence and presence of S9 mix was 3435.5 μg/mL. In the absence and presence of S9 mix RTG was reduced to 39 and 48% respectively. In the 24 hour treatment, the maximum concentration assessed for mutant frequency was

1750 μg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The RTG was reduced to 12%. In all tests the concurrent vehicle and positive control were within acceptable ranges.

Conclusions:
OS 1600 did not demonstrate mutagenic potential in this 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 OS1600 in accordance with OECD Guideline 476. Based on a preliminary test, cultures were exposed up to 3435.5 µg/mL for 3 h with and without metabolic activation and for 24 h without metabolic activation. Following a 3 h exposure, relative suspension growth (RSG) was reduced from 142 to 30% and from 123 to 59% in the absence and presence of S9 mix respectively. Following a 24 h exposure in the absence of S9 mix RSG was reduced from 131 to 1%. Following 3 h treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. In the absence and presence of S9 mix RTG was reduced to 39 and 48% respectively. In the 24 hour treatment, no increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The RTG was reduced to 12%. In all tests the concurrent vehicle and positive control were within acceptable ranges. It was concluded that OS 1600 did not demonstrate mutagenic potential in this 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: An in-vivo micronucleus test in Crl: CD(SD) rats was performed with OS1600 according to OECD Guideline 474. Based on preliminary results, 6 -8 male rats per group were treated with 0 (control), 125, 250 and 500 mg/kg bw orally by gavage (2 mL/kg) on two occasions approximately 24 hours apart. Bone marrow smears were obtained 24 hours after administration of the second dose. It was concluded that OS 1600 did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male rats when administered orally by gavage in this in vivo test procedure.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
All animals used on this study were Crl: CD(SD) rats and were sourced from Charles River UK Limited, Margate, Kent, England. Animals
used on the study weighed between:

Preliminary toxicity test: Males weighed between 194g to 205g.
Females weighed between 164g to 181g.
Micronucleus test: Males weighed between 174g to 224g

Animal age on despatch and on Day 1 of dosing were:

Preliminary toxicity test: On despatch Males and females ca 42 days old.
Day 1 Males and females ca 48 days old.
Micronucleus test: On despatch Males ca 42 days old.
Day 1 Males ca 49 days old.

Each group was kept, with the sexes separated, in cages and maintained in a controlled environment, with the thermostat and relative humidity target ranges set at 19 to 23°C and 40 to 70% respectively. Temperature and humidity were within range throughout the study. The room was
illuminated by artificial light for 12 hours per day. All animals were allowed free access to pelleted expanded rat and mouse No.1 maintenance
diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) and tap water ad libitum.

Food, chew blocks and tap water are routinely analysed for quality at source. All animals were given small soft white untreated wood (ASPEN) chew blocks and a red plastic shelter for environmental enrichment, were acclimatised for a minimum of 5 days, examined daily and weighed prior to dosing.
Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
Stability and homogeneity of the test substance and of the test substance in the vehicle were not determined in this test and remain the responsibility of the Sponsor. Chemical analysis of dosing formulations for achieved concentration was not performed in this study.
Suspensions of the test substance were prepared in Corn oil obtained from Sigma, batch number MKBF6012V and MKBG9425V.

Cyclophosphamide obtained from Sigma, batch number 120M1253V was used as the positive control compound. A solution was prepared using purified water at a concentration of 2 mg/mL just prior to administration.

All animals were dosed orally by gavage. The vehicle control and test substance groups were dosed using a dose volume of 2 mL/kg. The positive control group were dosed using a dose volume of 10 mL/kg.
Duration of treatment / exposure:
2 consecutive days
Frequency of treatment:
daily for 2 consecutive days
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 males at 125 mg/kg/day
6 males at 250 mg/kg/day
8 males at 500 mg/kg/day (due to toxicity)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (single dose at 20 mg/kg/day, 2 mg/mL)
Tissues and cell types examined:
femur bone marrow
Details of tissue and slide preparation:
The femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of the femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.

The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976).

Fixation and slide staining:
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in cold tap water for 2 minutes
6 Stored at room temperature until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water

Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.
Evaluation criteria:
A positive response is normally indicated by a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes for the treatment group compared with the vehicle control group (p<0.01); individual and/or group mean values should exceed the laboratory historical control range (Morrison and Ashby 1995).
A negative result is indicated where individual and group mean incidences of micronucleated polychromatic erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent vehicle control group and where these values fall
within the historical control range.
An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant decrease in the proportion of polychromatic erythrocytes (p<0.01).
Statistics:
For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3, then on Groups 1 and 2. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
For micronucleated polychromatic erythrocytes at 24 hours, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3. Also, exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
Statistical significance was declared at the 1% level for all tests.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 and 1000 mg/kg bw/day
- Animals: 2 rats per sex and per dose
- Clinical signs of toxicity in test animals:
At 500 mg/kg bw/day: unsteady gait, piloerection, underactive behaviour and hunched posture, hindlimbs (females). All animals survived until scheduled termination on Day 3. No net loss in bodyweight was observed.
At 1000 mg/kg bw/day: Unsteady gait, piloerection, underactive behaviour, splayed hindlimbs (males), prostrate posture, slow breathing, shallow breathing (males), deep breathing (males) and unresponsive behaviour (males), overactive behaviour (females), uncoordinated gait (females) and hunched posture (females). Animals were killed in extremis on Day 1, 1.5 and 2.5 hours post dose due to the severity of the clinical signs observed, consequently the maximum tolerated dose had been exceeded. The post mortem examinations did not find any signs of mis-dosing

RESULTS OF DEFINITIVE STUDY
- Micronucleated polychromatic erythrocyte counts (MPCE): No statistically significant increases in the number of MPCE in male animals. Cyclophosphamide caused a statistically significant increase in the frequency of MPCE (p<0.01) in male animals.
- Micronucleated normochromatic erythrocytes (MNCE): No significant increases in the incidence of MNCE in male animals.
- Proportion of polychromatic erythrocytes (%PCE): No statistically significant decreases in the proportion of PCE in male animals. Cyclophosphamide caused a statistically significant decrease in the proportion of PCE (p<0.01) in male animals.
- Clinical signs: At 250 mg/kg/day underactive behaviour, piloerection and unsteady gait were observed. At 500 mg/kg/day unsteady gait, underactive behaviour, piloerection, hindlimbs splayed, rales/noisy breathing and hunched posture were observed. Bodyweight loss of 15% from Day 2 to Day 3 was observed in one animal administered 500 mg/kg/day test item.
Conclusions:
OS 1600 did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD(SD) rats when administered orally by gavage in this in vivo test procedure.
Executive summary:

An in-vivo micronucleous test in Crl: CD(SD) rats was performed with OS1600 according to OECD Guideline 474. Based on preliminary resutls, 6 -8 male rats per group were were treated with 0 (control), 125, 250 and 500 mg/kg bw orally by gavage (2 mL/kg) on two occasions approximately 24 hours apart. Bone marrow smears were obtained from animals in the vehicle control and in each of the test substance groups 24 hours after administration of the second dose. One smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes (MPCE). The proportion of polychromatic erythrocytes (%PCE) was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes (MNCE) was also kept. No statistically significant increases in the frequency of MPCE and no statistically significant decreases in the % PCE were observed at any treatment level, compared to vehicle control values. It was concluded that OS 1600 did not show any evidence of causing an increase in the induction of MPCE or bone marrow cell toxicity in male rats when administered orally by gavage in this in vivo test procedure.

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 OS1600 is not genotoxic.

Additional information

Short description of key information:

In vivo and in vitro genetic toxicology studies indicate that OS1600 is not genotoxic.

Justification for classification or non-classification

Based on the available experimental data, OS1600 was determined to be non-genotoxic, and therefore it is not classified in accordance with CLP Regulation (EC) no. 1727/2008.