Registration Dossier

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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

As described in this dossier, Sa 57 did not induce gene mutations in Salmonella typhimurium and Escherichia coli strains applying pre-incubation or plate incorporation up to 5000 µg/plate in a GLP compliant study according to OECD Technical Guidelines 471.
As part of the described tiered testing approach for the intended tonnage band increase, Sa 57 was investigated for chromosome aberrations in
peripheral human lymphocytes and for gene mutations in V79 Chinese hamster cells in the absence and presence of metabolic activation in two GLP compliant studies according to OECD Technical Guidelines 473 and 476 respectively.
Sa 57 turned out to be not clastogenic even though an increased number of polyploid cells was observed in the absence of S9-mix, at the 48 h
continuous exposure time at the highest concentration tested. The latter might indicate a potential of Sa 57 to disturb mitosis. However, the absence of metabolic activation does not reflect the in vivo situation properly. In the gene mutation study, Sa 57 did increase the mutant frequency at the HPRT locus in a biologically relevant manner in the absence and presence of metabolic activation. However, the results are questionable because the observed increases in mutant frequency were not dose dependent.
Thus, in conclusion Sa 57 is not considered clastogenic even though some uncertainty remains with regard to its potential ability to disturb mitotic
processes. There are conflicting data concerning the gene mutation potential. In an assay with bacteria, Sa 57 did not cause gene mutations while in
human lymphocytes mutant frequency was increased, though without clear dose-response relationship.

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S 9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
DMF
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100 (10µg/plate), without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA 1537 (50 µg/plate), TA 98 (10 µg/plate), without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA (3 µL/plate), without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100 (2.5 µg/plate), WP2 uvr A (10µg/plate)
Details on test system and experimental conditions:
The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus".
In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
Strain WP2 (4) and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a
repair-deficient strain may be more readily mutated by agents.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY:
The S. typhimurium and E. coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

EVALUATION OF RESULTS:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
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

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains with and without metabolic activation. Only in experiment II a minor reduction in the number of revertants (below the indication factor of 0.5), occurred in strain WP2 uvrA at 5000 µg/plate in the absence of metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with SAT 090073 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, SAT 090073 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of SAT 090073 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains with and without metabolic activation. Only in experiment II a minor reduction in the number of revertants (below the indication factor of 0.5), occurred in strain WP2 uvrA at 5000 µg/plate in the absence of metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with SAT 090073 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10.06.2013 - 25.08.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Principles of method if other than guideline:
Deviations:
- Source of S9 mix, no influence on the quality or integrity of the study.
- Humidity, no effect on the results of the study.
- No. of metaphases: In the second cytogenetic assay in one of the duplicate cultures of the positive control (24 h exposure in the absence of S9-mix and 3 h exposure in the presence of S9-mix) only 86 and 88 metaphases were examined for chromosome aberrations.
Evaluation: No more scorable metaphases were present an the slides. A statistically significant increase in the number of cells with chromosome aberrations was observed in the scored metaphases of the positive control. Scoring of additional metaphases would have given limited additional information and would not have any effect an the outcome of the study.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Cultured peripheral human lymphocytes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix
Test concentrations with justification for top dose:
FIRST CYTOGENETIC ASSAY / DOSE RANGE FINDING STUDY:
- 10, 33, 100 µg/ml (3h exposure, with and without S9-mix)
- 1, 3, 10, 33, 100, 333, 1000 µg/ml (24h and 48h exposure, without S9-mix)

SECOND CYTOGENETIC ASSAY:
- 3, 10, 33, 100, 150, 200, 250, 300 µg/ml (24h exposure, without S9-mix)
- 0.3, 1, 3, 10, 33, 100, 200 µg/ml (48h exposure, without S9-mix)
- 10, 33, 100 µg/ml (3h exposure, with S9-mix)
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Untreated negative controls:
other: vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Mitomycin C without S9-mix / Cyclophosphamide with S9-mix
Evaluation criteria:
One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was >=25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) lt induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, onesided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present an the slides should not interfere with the scoring of chromosome aberrations.
Statistics:
Chi-square test, one-sided
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: vehicle control served as negative control
Positive controls validity:
valid

DOSE RANGE FINDING TEST / FIRST CYTOGENETIC ASSAY

 

At a concentration of 100 μg/ml Sa 57 precipitated in the culture medium. In the dose range finding study, at the 3 h exposure time, blood cultures were treated in duplicate with 10, 33 and 100 μg/ml culture medium with and without S9-mix (first cytogenetic assay).

At the 24 h and 48 h continuous exposure time single blood cultures were treated with 1, 3, 10, 33, 100, 333 and 1000 μg/ml culture medium without S9-mix. The test substance was tested beyond the limit of solubility to obtain adequate toxicity data.

 

Both in the absence and presence of S9-mix, Sa 57 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Table 1-2).

Both in the absence and presence of S9-mix, Sa 57 did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

 

SECOND CYTOGENETIC ASSAY

 

To obtain more information about the possible clastogenicity of Sa 57, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Sa 57 in the absence of S9-mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Sa 57.

The following dose levels were selected for the second cytogenetic assay:

 Without S9-mix:

- 3, 10, 33, 100, 150, 200, 250 and 300 μg/ml culture medium (24 h exposure time, 24 h fixation time).

- 0.3, 1, 3, 10, 33, 100 and 200 μg/ml culture medium (48 h exposure time, 48 h fixation time).

 With S9-mix:

- 10, 33 and 100 μg/ml culture medium (3 h exposure time, 48 h fixation time).

 

Based on the observations the following doses were selected for scoring of chromosome aberrations:

 Without S9-mix:

- 3, 200 and 250 μg/ml culture medium (24 h exposure time, 24 h fixation time).

- 1, 33 and 100 μg/ml culture medium (48 h exposure time, 48 h fixation time).

 With S9-mix:

- 10, 33 and 100 μg/ml culture medium (3 h exposure time, 48 h fixation time).

 

Both in the absence and presence of S9-mix, Sa 57 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Table 3-5).

Both in the absence and presence of S9-mix, Sa 57 did not increase the number of cells with endoreduplicated chromosomes. However, Sa 57 increased the number of polyploid cells in the absence of S9-mix, at the 48 h continuous exposure time.

 

EVALUATION OF THE RESULTS

 

The ability of Sa 57 to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h exposure time) or on toxicity,

inhibition of the mitotic index of about 50% or greater (24 h and 48 h continuous exposure time).

Duplicate cultures are indicated by A and B.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range.

The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. lt was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Both in the absence and presence of S9-mix Sa 57 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of Sa 57 an the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Sa 57 does not disturb cell cycle progression. However, it was noted that Sa 57 increased the number of polyploid cells in the absence of S9-mix, at the 48 h continuous exposure time at the highest concentration tested. This may indicate that Sa 57 has the potential to disturb mitotic processes.

 

 

TABLE 1 - FIRST CYTOGENETIC ASSAY

Chromosome aberrations (3 h exposure / 24 h fixation / without S9-mix)

 

 

DMSO

10 µg/ml

33 µg/ml

100 µg/ml

MMC
0.5 µg/ml

Culture

A

B

A

B

A

B

A

B

A

B

Mitotic index [%]

100

114

126

92

120

No. cells scored

100

100

100

100

100

100

100

100

100

100

Total aberrations (+ gaps)

0

1

0

2

3

4

0

0

25

25

Total aberrations (- gaps)

0

1

0

2

3

4

0

0

25

25

 

 

TABLE 2 - FIRST CYTOGENETIC ASSAY

Chromosome aberrations (3 h exposure / 24 h fixation / with S9-mix)

 

 

DMSO

10 µg/ml

33 µg/ml

100 µg/ml

CP
10 µg/ml

Culture

A

B

A

B

A

B

A

B

A

B

Mitotic index [%]

100

109

110

110

55

No. cells scored

100

100

100

100

100

100

100

100

50

50

Total aberrations (+ gaps)

1

1

1

0

1

0

0

0

32

34

Total aberrations (- gaps)

1

1

1

0

1

0

0

0

32

34

 

 

TABLE 3 - SECOND CYTOGENETIC ASSAY

Chromosome aberrations (24 h exposure / 24 h fixation / without S9-mix)

 

 

DMSO

3 µg/ml

200 µg/ml

250 µg/ml

MMC-C
0.2 µg/ml

Culture

A

B

A

B

A

B

A

B

A

B

Mitotic index [%]

100

68

68

51

40

No. cells scored

100

100

100

100

100

100

100

100

100

86

Total aberrations (+ gaps)

0

1

1

0

0

0

1

0

50

48

Total aberrations (- gaps)

0

1

1

0

0

0

1

0

49

48

 

 

TABLE 4 - SECOND CYTOGENETIC ASSAY

Chromosome aberrations (48 h exposure / 48 h fixation / without 9-mix)

 

 

DMSO

1 µg/ml

33 µg/ml

100 µg/ml

MMC-C
0.1 µg/ml

Culture

A

B

A

B

A

B

A

B

A

B

Mitotic index [%]

100

84

67

59

62

No. cells scored

100

100

100

100

100

100

100

100

50

50

Total aberrations (+ gaps)

0

1

2

0

0

1

0

1

37

47

Total aberrations (- gaps)

0

1

2

0

0

0

0

1

36

47

 

 

TABLE 5 - SECOND CYTOGENETIC ASSAY

Chromosome aberrations (3 h exposure / 48 h fixation / with S9-mix)

 

 

DMSO

10 µg/ml

33 µg/ml

100 µg/ml

CP
10 µg/ml

Culture

A

B

A

B

A

B

A

B

A

B

Mitotic index [%]

100

73

52

48

*

No. cells scored

100

100

100

100

100

100

100

100

100

100

Total aberrations (+ gaps)

2

0

2

1

0

0

0

0

50

32

Total aberrations (- gaps)

2

0

2

0

0

0

0

0

49

30

 

* CP fixed after 24 h, therefore no calculation of the mitotic index as percentage of control

 

Conclusions:
It is concluded that this test is valid and that Sa 57 is not clastogenic in human lymphocytes under the experimental conditions described in this report. Sa 57 may have the potential to disturb mitotic processes.
Executive summary:

SUMMARY

Evaluation of the ability of Sa 57 to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment).

This report describes the effect of Sa 57 an the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of Sa 57

was tested in two independent experiments.

The study procedures described in this report were based an the most recent OECD and EC guidelines.

Batch Sun-Sa-9940-57 of Sa 57 was a yellow hazy viscous liquid with a purity of >99%. Sa 57 was soluble in dimethyl sulfoxide at concentrations of 100 mg/ml and below but formed a suspension at the concentration of 331 mg/ml.

In the first cytogenetic assay, Sa 57 was tested up to 100 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Sa 57 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Sa 57 was tested up to 250 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 100 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-mix Sa 57 was also tested up to 100 μg/ml for a 3 h exposure time with a 48 h fixation time. Sa 57 precipitated in the culture medium at this dose level.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with

chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Sa 57 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No effects of Sa 57 an the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Sa 57 does not disturb cell cycle progression. However, it was noted that Sa 57 increased the number of polyploid cells in the absence of S9-mix, at the 48 h continuous exposure time at the highest concentration tested. This may indicate that Sa 57 has the potential to disturb mitotic processes.

Finally, it is concluded that this test is valid and that Sa 57 is not clastogenic in human lymphocytes under the experimental conditions described in this report. Sa 57 may have the potential to disturb mitotic processes.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July - September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Principles of method if other than guideline:
Deviations:
- The temperature was recorded to be outside the range of 37.0 ± 1.0°C as specified in the protocol for approximately 2.5 hour with a minimum of 32.9°C in the absence of S9-mix and for approximately 2 hours with a minimum of 33.5°C in the presence of S9-mix.
Evaluation: The deviation in the temperature was caused by adjustment of the temperature in the incubator after opening of the incubator door. Since the culture flasks were covered with a screw cap and a normal cell growth of the negative controls was observed and the spontaneous mutation frequency in the solvent controls was within the laboratory historical range, these deviations of the temperature have no effect on the results of the study.
- The cloning efficiency of the solvent controls in the part of the experiment with S9-mix (CE day7, presence of S9-mix) was not within the protocolled range (60 - 130%).
Evaluation: Although the values were above the limit of the range, the spontaneous mutation frequency in the solvent controls were within the laboratory historical range, therefore this deviation in the absolute cloning efficiency has no effect on the results of the study .
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix
Test concentrations with justification for top dose:
DOSE RANGE FINDING TEST:
- 0.3, 1, 3, 10, 33, 100 and 333 µg/ml (with and without S9-mix),

MUTATION EXPERIMENT:
- 0.1, 0.3, 1, 3, 6.6, 10, 12.5, 16, 20 and 25 µg/ml (without S9-mix),
- 0.3, 1, 3, 10, 33, 100, 130, 160, 200 and 250 µg/ml (with S9-mix)
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Untreated negative controls:
other: vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Remarks:
Ethylmethanesulphonate without S9-mix / 3-Methylcholanthrene with S9-mix
Evaluation criteria:
DETERMINATION OF MUTANT COLONIES
A mutation assay is considered acceptable if it meets the following criteria:
a) The absolute colony forming efficiency of the solvent controls should be between 60 and 130%
b) At least seven doses of the test substance should have an acceptable number of surviving cells which could be analysed for expression of the HPRT mutation.
c) The spontaneous mutant frequency in the solvent-treated control is <10 per 100000 clonable cells.
d) The positive control substances induced significant (at least three-fold) increases in the mutant frequency.
e) The selected dose range has to include a clearly toxic concentration (10 to 30%) of the average of solvent controls or should exhibit limited solubility or should extend to 5 mg/plate.
lf one of the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test will be rejected and repeated.

DATA EVALUATION
In addition, 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. No formal hypothesis testing is done.
A test substance is considered positive (mutagenic) in the mutation assay if it induces at least a three-fold increase in the mutation frequency compared to the solvent control 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 show a mutation frequency of at least three-fold compared to the solvent control.
b) The results are confirmed in an independently repeated test.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: vehicle control served as negative control
Positive controls validity:
valid

SOLUBILITY

 

Sa 57 precipitated in the exposure medium at concentrations of 100 μg/ml and above. Sa 57 was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 μg/ml.

 

DOSE RANGE FINDING TEST

 

In the dose range finding test, V79 Chinese hamster cells were treated with a test substance concentration range of 0.3 to 333 μg/ml in the absence and presence of S9-mix with a 4 hour treatment period. Table 1 shows the relative cell survival as the percentage cloning efficiency in the absence and presence of S9-mix with a 4 hour treatment period with various concentrations of Sa 57.

No cell survival in the cloning efficiency was observed at test substance concentrations of 33 μg/ml and above in the absence of S9-mix and at 333 μg/ml in the presence of S9-mix.

 

MUTATION EXPERIMENT

 

Table 2 shows the percentages of cell count 24 hours after start of the treatment, the cloning efficiency of Day 0 and Day 7and the mutation frequencies for various concentrations of Sa 57.

 

Based on the results of the dose range finding test the following dose levels were selected for the mutation experiment:

- Without S9-mix: 0.1, 0.3, 1, 3, 6.6, 10, 12.5, 16, 20 and 25 μg/ml

- With S9-mix: 0.3, 1, 3, 10, 33, 100, 130, 160, 200 and 250 μg/ml

In the absence of S9-mix, the dose levels of 20 and 25 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 0.3 to 100 μg/ml showed no cytotoxicity. Therefore, the dose levels of 0.3 and 1 μg/ml were not regarded relevant for mutation frequency measurement.

 

The dose levels selected to measure mutation frequencies at the HPRT-locus were:

- Without S9-mix: 0.1, 0.3, 1, 3, 6.6, 10, 12.5 and 16 μg/ml

- With S9-mix: 3, 10, 33, 100, 130, 160, 200 and 250 μg/ml

 

Evaluation of mutagenicity:

In the absence of S9-mix, SA 57 induced a 6.8-fold increase in the mutant frequency in the mutation frequency at the HPRT locus.

In the presence of S9-mix SA 57 induced a 5.6-fold increase in the mutant frequency in the mutation frequency at the HPRT locus at the dose level of 100 μg/ml.

 

DISCUSSION

 

The spontaneous mutation frequencies in the solvent-treated control cultures were within the historical control data range.

 

Mutation frequencies in cultures treated with positive control chemicals were increased by 14-fold for EMS in the absence of S9-mix, and 6.4-fold for 3-MCA in the presence of S9-mix. lt 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.

 

Sa 57 induced 6.8- and 5.6-fold increases in the mutant frequency at the HPRT locus in the absence and presence of S9-mix, respectively. Although the observed increases were not doserelated, the increases were outside the historical control data range and more than 3-fold.

Therefore, these increases are considered to be biologically relevant and Sa 57 is considered to be mutagenic in the absence or presence of S9-mix.

 

 

TABLE 1 - DOSE RANGE FINDING TEST

Cloning efficiency (rel. survival, % of control) of Sa 57 in V79 Chinese hamster cells

 

Dose [µg/ml]

Without S9-mix

With S9-mix

DMSO (control)

100

100

0.3

123

103

1

112

116

3

119

122

10

99

113

33

0

119

100*

0

109

333*

0

0

 

 

TABLE 2 - MUTAGENICITY TEST

Cytotoxic and mutagenic response of Sa 57 in V79 / HPRT test system

 

Dose [µg/ml]

Cell count day2, 24h after treatment,
% of controls

CE day0,
% of controls

CE day7, absolute %

Mean no. of mutants / plate

MF x 105

Without S9-mix, 4h treatment

DMSO (control 1)

100

100

117

1.3

1.1

DMSO (control 2)

-

-

117

0.9

0.8

0.1

10

101

124

0.6

0.5

0.3

97

107

135

0.1

0.1

1

97

84

132

2.4

1.8

3

92

99

111

3.9

3.6

6.6

91

97

118

1.3

1.1

10

90

89

116

4.0

3.4

13

50

80

102

6.6

6.5

16

25

38

110

3.9

3.5

EMS(pos. control)

66

93

94

12.2

13.1

With S9-mix, 4h treatment

DMSO (control 1)

100

100

153

2.1

1.4

DMSO (control 2)

-

-

174

1.2

0.7

3

125

110

121

4.1

3.4

10

125

105

206

2.3

1.1

33

133

108

90

3.6

4.0

100*

104

104

92

5.7

6.1

130*

66

99

94

5.1

5.4

160*

97

113

99

5.8

5.9

200*

61

51

173

2.2

1.3

250*

12

1

126

2.9

2.3

3-MCA (pos. control)

72

109

95

6.4

6.7

 

* Sa 57 precipitated in the exposure medium

Conclusions:
lt is concluded that Sa 57 is mutagenic in the gene mutation test with V79 Chinese hamster cells under the experimental conditions described in this report.
Executive summary:

SUMMARY

Evaluation of the mutagenic activity of Sa 57 in an in vitro mammalian cell gene mutation test with V79 Chinese hamster cells.

This report describes the effects of Sa 57 on the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in V79 Chinese hamster cells. The test was performed in in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch Sun-Sa-9940-57 of Sa 57 was a yellow hazy viscous liquid with a purity of >99%. The test substance was dissolved in dimethyl sulfoxide.

Sa 57 was tested up to concentrations of 16 and 250 μg/ml in the absence and presence of S9-mix. The incubation time was 4 hours. Appropriate toxicity was observed at these dose levels in the absence and presence of S9-mix.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased 14-fold for EMS in the absence of S9-mix, and by 6.4-fold for 3-MCA in the presence of S9-mix. lt was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

Sa 57 induced 6.8- and 5.6-fold increases in the mutant frequency at the HPRT locus in the absence and presence of S9-mix, respectively. Although the observed increases were not dose-related, the increases were outside the historical control data range and more than 3-fold.

Therefore, these increases are considered to be biologically relevant and Sa 57 is considered to be mutagenic in the absence or presence of S9-mix.

lt is concluded that Sa 57 is mutagenic in the gene mutation test with V79 Chinese hamster cells under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In order to finally clarify the genotoxic potential, an in vivo combined micronucleus and comet assay was conducted according to OECD TG 474 and 489. In the respective micronucleus test Sa 57 was not clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of 2000 mg/kg under the experimental conditions. Furthermore, in the comet assay Sa 57 did not provoke DNA damage in the Comet assay in liver and glandular stomach cells under the experimental conditions.

In conclusion Sa 57 is not considered to have a genotoxic potential in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: Combined DNA damage and/or repair and chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 489 (Alkaline Comet Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: combined Comet and Micronucleus assay
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar rats (outbred, SPF-Quality) are used as test system. These rats are recommended by international guidelines (e.g. EPA, FDA, OECD, EC).
Sex:
male
Details on test animals or test system and environmental conditions:
ANIMALS
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).
Wistar WI (Han) rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany.
Young adult animals were selected (6-7 weeks old at the start of treatment). The total number of animals used in the dose-range finding study was 3 and in the main study 30. In the main study 5 male rats were treated per sampling time in each treatment group. Six additional animals were used for blood sampling for bioanalysis. These animals were treated with vehicle or the highest test item concentration (3 animals per group).
The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. The body weights of the rats at the start of the treatment with Sa 57 were within 20% of the sex mean. The mean body weight was 151 (± 7.4 g) and the range was 135 – 186 g. The rats were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups. The acclimatization period was at least 6 days before the start of treatment under laboratory conditions. On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.


CONDITIONS
A controlled environment was maintained in the room with optimal conditions of approximately 10 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 19.2 – 21.8°C), a relative humidity of 40 - 70% (actual range: 37 - 68%) and a 12 hour light/12 hour dark cycle. Due to e.g. cleaning procedures, temporary deviations from the humidity (maximum 3%) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Accommodation: Group housing of maximum 5 animals per sex in labeled Macrolon cages (type MIV height 180 mm, length 600 mm and width 330 mm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

Diet: The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.

Water: The animals had free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures.. There were no findings that could interfere with the study.
Route of administration:
oral: gavage
Vehicle:
Corn oil (Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands)

No correction was made for the purity/composition of the test item. Sa 57 was suspended (2000 and 1000 mg/kg group) or dissolved (500 mg/kg group) in corn oil. The specific gravity of corn oil is 0.9 g/mL. Sa 57 concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension or to dissolve the test item (for a maximum of 17 minutes at 28ºC in the main study). Sa 57 concentrations were dosed within 4 hours after preparation.
Details on exposure:
The rats were dosed for three consecutive days (once daily) using an oral intubation with a plastic gavage needle of a high, an intermediate and a low dose of Sa 57. The rats were dosed twice with the positive control EMS and once with CP. A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat).

The first dose of the test item and vehicle was administered at t = 0 h. The second and third dose will be administered at t=24 h and t= 45, respectively. The positive control CP was administered once at t = 0 h and EMS was administered at t=24 and t=45. The animals were sacrificed at approximately t = 48 h by abdominal aorta bleeding under isoflurane anesthesia.

Animals were checked for mortality at least twice a day. The time of death was recorded as precisely as possible. The systemic toxic signs were recorded at least once a day from start of treatment onwards. All signs were recorded. The animals were weighed just prior to dosing.
Duration of treatment / exposure:
3 d
Frequency of treatment:
once daily for three consecutive days
Post exposure period:
- Dose-range Finding study: 1-4 days (mortality and physical condition were recorded at least once a day)
- Main study: Sacrifice on day 3
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control; Group A and A'
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
Group B and B'
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group C
Dose / conc.:
500 mg/kg bw/day
Remarks:
Group D
No. of animals per sex per dose:
5 (plus 3 additional animals for vehicle control and highest dose group)
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control for the micronucleus test was Cyclophosphamide (CP; CAS no. 50-18-0) at 20 mg/kg body weight dissolved in physiological saline. The stock solutions of CP were stored in aliquots at ≤ -15°C in the dark and one sample was thawed immediately before use. The route of administration was oral and the dosing volume was 10 mL/kg body weight.

The positive control for the Alkaline Comet test was Ethyl Methanesulfonate (EMS; CAS no. 62-50-0) at 200 mg/kg body weight dissolved in physiological saline. EMS was used within 3 hours after preparation and the route of administration was oral. The dosing volume was 10 mL/kg body weight.
Tissues and cell types examined:
The bone marrow was examined for micronuclei while the Comet part of the combined assay was conducted with liver tissue and (glandular) stomach cells.
Blood was collected from the six satellite animals (high dose group and vehicle control group) for bioanalysis.
Details of tissue and slide preparation:
BONE MARROW SMEARS FOR MICRONUCLEI
The supernatant was removed with a Pasteur pipette. Approximately 500 µL serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, the Netherlands). This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands)

COMET SLIDES
To 20 µL of the cell suspension, 280 µL melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added. The cells were mixed with the LMAgarose and
50 µL was layered on a precoated Comet slide (Trevigen) in duplicate. Three slides per tissue were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 10-42 minutes in the refrigerator in the dark until a clear ring appeared at the edge of the Comet slide area.
The cells on the slides were overnight (approximately 16-17 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 29-32 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1 Volt/cm for liver and at 0.7 Volt/cm for stomach. The electrophoresis was performed for 30 minutes under constant cooling (actual temperature 4.0 °C) for liver and for 20 minutes under constant cooling (actual temperature 4.0 °C) for stomach. After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in absolute ethanol (Merck, Darmstadt, Germany) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark.

Evaluation criteria:
see "Any other information on materials and methods"
Statistics:
Student's t test; Dunnett's test; ToxRat Professional v 3.2.1
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
micronucleus test
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
comet assay
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

DOSE RANGE FINDING STUDY

In a dose-range finding study 3 male animals were dosed via oral gavage with 2000 mg/kg body weight of SA 57. One animals showed no treatment related clinical signs after the first and second dosing. Within approximately one hour after the third dosing this animal was lethargic and within two hours after the third dosing this animal showed in addition ataxia. The animal appeared recovered within 20-21 hours after the third dosing. The other two animals were lethargic and had diarrhea within approximately 1 hour after the first dosing. The animals appeared recovered within approximately 22 hours after the first dosing. Within approximately 1 hour after the second dosing the animals were lethargic. The animals appeared recovered within approximately 22 hours after the second dosing but the animals did not completely eat the limited amount of food that was provided for them overnight. Within approximately 1 hour after the third dosing the animals were lethargic and showed ataxia. The severity of the effects was generally considered to be slight.

MAIN STUDY

Based on the results of the dose-range finding study dose levels of 500, 1000 and 2000 mg/kg body weight were selected as appropriate doses for the main study.

Mortality and Toxic Signs

The animals of the group treated with 500 mg Sa 57/kg bw and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. The following clinical observations were made in the group treated with 1000 mg Sa 57 /kg bw: The animals showed no treatment related clinical signs after the first and second dosing. Within approximately 1 hour after the third dosing 3 of the 5 animals were lethargic. The other two animals showed no treatment related clinical signs.

The following clinical observations were made in the group treated with 2000 mg Sa 57 /kg bw: Within approximately the first hour after the first dosing all animals were lethargic. In addition one animal showed ataxia. Within approximately one hour after the second dosing all animals were lethargic and showed ataxia. All animals appeared recovered within approximately 19 hours after the second dosing. Within approximately one hour after the third dosing all animals were lethargic and showed ataxia. The severity of the effects was generally considered to be slight.

Micronucleated polychromatic erythrocytes

The mean number of micronucleated polychromatic erythrocytes per group and the mean ratio of polychromatic to normochromatic erythrocytes are presented in the table below. The mean number of micronucleated polychromatic erythrocytes scored in Sa 57 treated groups were compared with the corresponding solvent control group. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of Sa 57 treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

Ratio of polychromatic to normochromatic erythrocytes

The animals of the groups which were treated with Sa 57 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test item on erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

 Group  Dose [mg/kg bw]  Mean no. polychromatic erythrocytes  Mean ratio polychrom. / normochrom. erythrocytes
 A vehicle control  0  5.0  0.99
 B Sa 57  2000  2.6  0.88
 C Sa 57  1000  2.4  0.99
 D Sa 57  500  3.4  1.09
 F pos. control CP  20  40.4  0.46

Comet Slide Analysis

After treatment, single cell suspensions from the liver and stomach were prepared. The viability of one single cell suspension per tissue per group was assessed by using trypan blue. The viability of all single suspension was 100%. Comet slides were prepared and analyzed.

- Liver: No statistically significant increase in the mean Tail Intensity (%) was observed in liver cells of Sa 57-treated male animals at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity in liver cells of male vehicle-treated rats was 4.13%. All values were within the historical data control range. EMS, the positive control item, showed a Mean Tail Intensity of 97.96%. EMS treatment induced a statistically significant increase of 23.7-fold (p<0.001 Student’s t test) in the mean Tail Intensity (%) in liver cells of male rats when compared to the vehicle. Hence, the acceptability criteria of the test were met.

- Glandular Stomach: No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells of Sa 57-treated male animals at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity in stomach cells of male vehicle-treated rats was 43.05%. All values were within the historical data control range. EMS, the positive control item, showed a Mean Tail Intensity of 88.47%. EMS treatment induced a statistically significant increase of 2.1-fold (p<0.001 Student’s t test) in the mean Tail Intensity (%) in stomach cells of male rats when compared to the vehicle. Hence, the acceptability criteria of the test were met. A decrease in Tail Intensity was observed at 1000 mg/kg in glandular stomach. Since this decrease was only observed at 1000 mg/kg and thus not at 500 and 2000 mg/kg (no dose related effect) and also not in liver this decrease is considered biologically not relevant and caused by variation in the data. Moreover a decrease does not indicate a genotoxic effect and overall there is thus no effect on the study conclusion.

 Group  Dose [mg/kg bw]  Tail Intensity [%] liver cells  Tail Intensity [%] stomach cells
 A vehicle control  0  4.13  43.05
 B Sa 57  2000  5.10  41.23
 C Sa 57  1000  4.12  17.26
 D Sa 57  500  2.73  50.18
 E pos. control EMS  200  97.96  88.47

Chemical analysis of dose preparations

The formulations of the second dosing of the main study were analyzed for concentration, homogeneity and stability.

- Concentration: The concentrations analyzed in the formulations of Group D (500 mg/kg bw), Group C (1000 mg/kg bw) and Group B (2000 mg/kg bw) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The mean accuracies were 100, 100 and 102% for Group D, C, B, respectively. No test item was detected in the Group A (vehicle control) formulation.

- Homogeneity: The formulations of Group D (500 mg/kg bw) and Group B (2000 mg/kg bw) were homogeneous (i.e. coefficient of variation ≤ 10%). The homogeneity (coefficient of variation %) was 3.4 and 5.0% for group D and B, respectively.

- Stability: Formulations of Group D (500 mg/kg bw) and Group B (2000 mg/kg bw) were stable when stored at room temperature under normal laboratory light conditions for at least 4 hours (i.e. mean concentrations after storage ± 10%). The relative difference between the mean concentration of the stability samples and six samples at t=0 taken at 10%, 50% and 90% height was -0.91 and -1.2% for group D and B, respectively.

Conclusions:
It is concluded that the micronucleus test was valid and that Sa 57 is not clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of 2000 mg/kg under the experimental conditions described in this report. Moreover, the comet assay was valid and Sa 57 does not provoke DNA damage in the Comet assay in liver and glandular stomach cells under the experimental conditions described in this report.
Executive summary:

Sa 57 was tested in a combined Micronucleus and Comet test in the rat.

The objective of the study was to obtain information on the potential genotoxicity of Sa 57 when administered to rats at a maximum required acute dose, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in liver and glandular stomach tissue.

The study procedures described in this report are in compliance with the most recent OECD and EC guidelines.

Batch SS-Sa-11074-80 of SA 57 was a yellow viscous liquidwith a purity of 99.3%. The test item was suspended (2000 mg/kg group and 1000 mg/kg group) or dissolved (500 mg/kg group) in corn oil.

In a dose range finding study, 3 male animals were dosed via oral gavage with Sa 57 at 2000 mg/kg/day body weight for three consecutive days. The treatment related clinical signs of the three animals treated with 2000 mg/kg/day were slight and comprised lethargy, ataxia, diarrhea (2/3 animals) and the limited amount of feed provided overnight before dosing was not completely eaten (2/3 animals). Based on the results of the dose range finding study, a dose level of 2000 mg/kg body weight was selected as the highest dose level in the main study (the maximum recommended dose in accordance with current regulatory guidelines).

In the main study, groups of 5 male animals were dosed via oral gavage with vehicle or with 2000, 1000 and 500 mg Sa 57 per kg body weight per day for three consecutive days. A positive control group (5 male rats) for the comet assays was dosed once daily for two consecutive days by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight and a positive control group (5 male rats) for the micronucleus assay was dosed once by oral gavage with 20 mg cyclophosphamide (CP) per kg body weight. In addition, blood for bioanalysis of Sa 57 in plasma was collected from 3 satellite animals for the 2000 mg/kg group (highest dose group) and from three satellite animals for the vehicle control group.

 

The animals of the group treated with 500 mg Sa 57/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. The animals treated with 1000 mg Sa 57 /kg body weight showed no treatment related clinical signs with exception of 3 animals which were lethargic after the third dose. The following clinical observations were made in the group treated with 2000 mg Sa 57 /kg body weight: Within approximately the first hour after the first dosing all animals were lethargic. In addition one animal showed ataxia. Within approximately one hour after the second dosing all animals were lethargic and showed ataxia. All animals appeared recovered within approximately 19 hours after the second dosing. Within approximately one hour after the third dosing all animals were lethargic and showed ataxia.The severity of the effects was generally considered to be slight.

 

Approximately 3-4 hours after the third dose of the vehicle or Sa 57, liver and glandular stomach tissue were collected for the comet assay. The animals were sacrificed by abdominal aorta bleeding under isoflurane anaesthesia. Single cell suspensions were made followed by comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed. Bone marrow smears were prepared for micronucleus analysis.

Bone marrow smears were analysed. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with
Sa 57 compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control item, induced a statistically significant 8.1-fold increase (Student's t test p<0.001) in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met. The groups that were treated with Sa 57 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

No statistically significant increase in the mean Tail Intensity (%) was observed in liver and glandular stomach cells of Sa 57-treated male rats at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity (%) in liver cells of vehicle treated male rats was 4.13%. All values were within the historical data control range. EMS, the positive control item, showed a mean Tail Intensity of 97.96%. EMS treatment induced a statistically significant increase of 23.7-fold (p<0.001 Student’s t test) in the mean Tail Intensity (%) in liver cells of male rats when compared to the vehicle treated rats. Hence, the acceptability criteria of the comet test in liver were met. The mean Tail Intensity (%) in glandular stomach cells of vehicle treated male rats was 43.05%. The positive control EMS, showed a mean Tail Intensity of 88.47% (2.1-fold statistically significant induction; Studentsttest p<0.001). The negative and positive control Tail Intensities were within the historical control data range. Hence, all criteria for an acceptable comet assay in stomach were met.

 

Formulation analysis was performed to determine the accuracy of preparation, homogeneity and stability of the test item in formulations. The concentrations analysed in the formulations of the high dose (2000 mg/kg), mid dose (1000 mg/kg) and low dose (500 mg/kg) were in agreement with target concentrations (i.e. mean concentrations of 102, 100 and 100%, respectively). No test substance was detected in the vehicle control. Overall it was concluded that the accuracy of preparation was acceptable. The formulations were homogeneous (i.e. coefficient of variation <5.0%) and stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

It is concluded that the micronucleus test was valid and that Sa 57 is not clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of 2000 mg/kg under the experimental conditions described in this report. Moreover, the comet assay was valid and Sa 57 does not provoke DNA damage in the Comet assay in liver and glandular stomach cells under the experimental conditions described in this report.

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

Additional information

Justification for classification or non-classification

Since Sa 57 was clearly negative in vivo in a combined micronucleus and comet assay conducted according to OECD TG 474 and 489, the classification criteria are not met.