Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: non mutagenic (OECD 471, GLP, K, rel. 1)

- CA/HL test: non clastogenic (OECD 473, GLP, K, rel. 1)

- hprt/CHO: non mutagenic (OECD 476, GLP, K, rel. 1)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1999-01-19 to 1999-02-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 471 and in compliance with GLP.
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)
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspection date: 1998-03-23
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene for S. thyphimurium and tryptophan gene for E.coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (10% S9-fraction from liver of male Sprague-Dawley rat injected with Aroclor 1254)
Test concentrations with justification for top dose:
- Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
- Mutation study, Experiment 1 & 2: 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: suspension observed with water at 50 mg/mL. Good solution with DMSO at 50 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
see Table 7.6.1/1
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
see Table 7.6.1/1
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposition duration: 48 hours

NUMBER OF REPLICATIONS: Triplicate plate per dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control for 1997).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 billion bacteria per mL.
4. Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.
Rationale for test conditions:
Tested up to the recommended maximum test concentration for soluble non-cytotoxic substances, i.e. 5000 µg/plate.
Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met: the test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnet's method of linear regression
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 5000 µg/plate in the presence of S9-mix only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES: The test material was toxic to TA100 at 5000 µg/plate, with S9-mix only, but was non toxic to TA100, without S9-mix, and E. coli strain WP2uvrA-, both with and without S9-mix. See Table 7.6.1/2.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. The comparison was made with the historical control ranges for 1997 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial lawn in all of the Salmonella tester strains at 5000 µg/plate in the presence of Aroclor-induced rat liver S9 only. No toxicity was observed in E. coli strain WP2uvrA-, either with or without S9. An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

Table 7.6.1/2 : Preliminary toxicity results

S9-mix

Strain

Dose (µg/plate

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

+

TA100

97

100

93

102

110

79

96

106

94

92

89PS

-

113

95

91

C

110

85

105

133

100

85

91P

+

WP2uvrA-

38

23

29

26

40

25

22

32

27

38

25P

-

29

37

31

38

33

18

32

28

27

28

34P

P = precipitate

S = sparse background lawn

C = contaminated

Mutation study: result tables are included in "Attached background material"

Conclusions:
The test item was not mutagenic both in the presence and absence of metabolic activation in S. thyphimurium strains TA1535, TA1537, TA98, TA100, and E.coli WP2 uvrA-.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E.coli strain WP2 uvrA- were exposed the test material diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the main tests was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial lawn in all of the Salmonella tester strains at 5000 µg/plate in the presence of Aroclor-induced rat liver S9 only. No toxicity was observed in E. coli strain WP2uvrA- , either with or without S9. An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

Under the test condition, ST 08 C 98 was not mutagenic with and without metabolic activation to S. thyphimurium strains TA1535, TA1537 TA98, TA100, and E.coli WP2 uvrA-.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2012-06-18 to 2012-09-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 476 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - 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
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspection date: 18-20 June 2012)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cells: CHO-KI cells were originally obtained from BIBRA. The cells are stored at -196°C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO).
- Type and identity of media:
Ham’s Nutrient Mixture F12, supplemented with 2 mM L glutamine and 50 µg/mL gentamicin. The resulting medium is referred to as H0.
H0 medium supplemented with 10% HiFCS referred to as H10, is used for general cell culture, e.g. when growing cells up from frozen stocks.
The selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-thioguanine (6-TG) at a final concentration of 10 µg/mL.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, assumed to be stable
- Periodically "cleansed" against high spontaneous background: yes, 4 days prior to exposure, spontaneous mutants were eliminated from the stock cultures by incubating the cells in H10 containing 15 µg/mL hypoxanthine, 0.3 µg/mL amethopterin and 4 µg/mL thymidine for three days.
All cell cultures are maintained at 37°C in a atmosphere of 5% CO2 in air.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (25% S9-fraction obtained from the liver of male Sprague-Dawley treated with Aroclor 1254 (500 mg/kg bw) by intraperitoneal route 5 days before dosing) (see Table 7.6.1/1)
Test concentrations with justification for top dose:
Preliminary toxicity test: 21.13, 42.25, 84.5, 169, 338, 676, 1352 and 2704 µg/mL (up to 10 mM).
Main test 1 (-S9 mix): 5, 10, 20, 40, 80, 120 and 140 µg/mL.
Main test 1 (+S9 mix): 5, 10, 40, 80, 140, 240, 340 and 400 µg/mL.
Additional test 1 (+S9 mix): 5, 10, 50, 200, 300, 400, 500, 600 and 700 µg/mL.
Main test 2 (-S9 mix): 5, 10, 20, 40, 60, 80 and 100 µg/mL.
Main test 2 (+S9 mix): 5, 40, 200, 300, 400, 500, 600 and 700µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (final concentration 1% v/v)
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance in a vehicle compatible with this test system was assessed. The test item was found to be soluble at 270.4 mg/mL (1M) in dimethyl sulphoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
250 µg/mL in DMSO
Positive control substance:
ethylmethanesulphonate
Remarks:
in the absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/mL in DMSO
Positive control substance:
3-methylcholanthrene
Remarks:
in the presence of S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
The cells were incubated for approximately 20 hours at 37°C, in an atmosphere of 5% CO2 in air, prior to exposure to the test substance on Day 1.
- Exposure duration: 3 hours.
- Expression time (cells in growth medium): 7 days, at 37°C, in a humidified atmosphere of 5% CO2 in air.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: duplicate cultures for each concentration of the test compound and positive controls, four individual cultures for solvent controls.

NUMBER OF CELLS EVALUATED: 10E06 cells from each culture were seeded to allow expression of the mutant phenotype.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (200 cells/plate)
Rationale for test conditions:
Tested up to cytotoxic concentrations.
Evaluation criteria:
The demonstration of a statistically significant increase in mutant frequency following exposure to the test substance;
Evidence of a relationship, over at least two dose levels, in any increase in mutant frequency;
Demonstration of reproducibility in any increase in mutant frequency;
The mean mutant frequency should fall outside the upper limit of the historical solvent control of 20 mutants per 10E6 survivors with a corresponding survival rate of 20% or greater.
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control to solvent control.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium were observed at 84.5 and 2704 µg/mL of more than 1.0 unit compared with the vehicle control.
- Effects of osmolality: The osmolality of the test substance in medium was tested at concentrations of 84.5 and 2704 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Evaporation from medium: not applicable
- Water solubility: not soluble in water
- Precipitation: No precipitate was seen at any level tested in bot tests in the absence of S9 mix. In test 1 (+S9 mix), precipitate was seen at 240 µg/mL and above post dosing and at the end of treatment. In additional test 1 (+S9 mix) and in test 2 (+S9 mix), precipitate was seen at 300 µg/mL and above post dosing and at the end of treatment.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity test, a four-hour exposure to 2704 µg/mL test item, in the absence or the presence of S9 mix, resulted in Day 1 relative survivals of 77 to 0 % and 45 to 0 % respectively. Precipitate was seen at 169 µg/mL and above at the end of treatment in the absence of S9 mix. Precipitate was seen at 169 µg/mL and above post dosing and at 338 µg/mL and above at the end of treatment in the presence of S9 mix. Concentrations for the main test were based upon these data.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Additional test 1 (+S9 mix): Although the mutant frequency of the concurrent solvent control is outside of the stated accepted range (20.50 mutants per 10E6 survivors) it is close to the historical maximum (20 mutants per 10E6 survivors) and therefore was considered to be acceptable.
- Additional test 1 (+S9 mix) and test 2 (+S9 mix): Several of the test item test cultures also had mutation frequencies that exceeded the historical control maximum. However, there was no dose-response relationship or statistical significance so these data were considered to be acceptable and not to be indicative of a positive response.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test 1 (-S9 mix): Exposure to test item resulted in Day 1 relative survivals of between 103 and 3 %. The Day 8 cloning efficiencies were reduced from 103 to 27 % relative to the solvent controls.
- Test 1 (+S9 mix): Exposure to test item resulted in Day 1 relative survivals of between 87 and 46 %. As an insufficient toxicity range was achieved an additional test was performed.
- Additional Test 1 (+S9 mix): Exposure to test item resulted in Day 1 relative survivals of between 108 to 21 % . The Day 8 cloning efficiencies were reduced from 115 to 93 % relative to the solvent control.
- Test 2 (-S9 mix): Exposure to test item resulted in Day 1 relative survivals of between 101 to 18 %. The Day 8 cloning efficiencies were reduced from 101 to 81 % relative to the solvent control.
- Test 2 (+S9-mix): Exposure to test item resulted in Day 1 relative survivals of between 93 to 19 %. The Day 8 cloning efficiencies were reduced from 90 to 79 % relative to the solvent control.

Table 7.6.1/2: Summary table

Test Article

Dose Level

µg/mL

Test 1

Test 2

3 hr Treatment ‑S9‑mix

3 hr Treatment +S9‑mix

3 hr Treatment ‑S9‑mix

3 hr Treatment +S9‑mix

Mean Relative Survival (%)

Mean Mutant Frequency (x106)

Mean Relative Survival (%)

Mean Mutant Frequency (x106)

Mean Relative Survival (%)

Mean Mutant Frequency (x106)

Mean Relative Survival (%)

Mean Mutant Frequency (x106)

DMSO

0

100

4.16

100

20.50

100

19.34

100

16.34

Romandolide

5

103

2.34

 

 

101

21.69

93

20.36

Romandolide

10

80

1.93

108

43.26

 

 

 

 

Romandolide

20

46

2.19

 

 

59

49.68

 

 

Romandolide

60

 

 

 

 

37

59.05

 

 

Romandolide

80

31

8.85

 

 

34

35.89

 

 

Romandolide

100

 

 

 

 

18

78.97

 

 

Romandolide

120

6

8.99

 

 

 

 

 

 

Romandolide

200

 

 

651

35.79

 

 

671

47.73

Romandolide

300

 

 

601

31.60

 

 

531

41.86

Romandolide

400

 

 

361

26.87

 

 

 

 

Romandolide

500

 

 

211

28.57

 

 

351

50.76

Romandolide

700

 

 

 

 

 

 

191

49.12

Ethyl methansulphonate

250

114

331.82**

 

 

56

274.92**

 

 

3-Methylcholanthrene

5

 

 

118

438.26**

 

 

107

463.09**

**   Statistically significant: p < 0.01

1   Precipitate observed by eye at the end of treatment

Conclusions:
The test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation, in both experiments.
Executive summary:

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, Chinese hamster ovary cells (CHO-K1) were exposed to the test item diluted in DMSO, in duplicate in the presence and absence of metabolic activation (S9-mix). The entire test was repeated to confirm the result of the first test. Three-hour exposures were used both with and without activation (S9) in both tests. The dose range of test material, plated out for expression of mutant colonies, was based on the results of a preliminary toxicity test, in which relative survivalvalues were from 77 to 0%, and 45 to 0%, after exposure to 21.13 to 2704 mg/mL in the absence and presence of S9 mix respectively (up to the maximum recommended level of10 mM).

In the absence of S9 mix, in Test 1, cells were exposed to concentrations from 5 to 140 µg/mL. Day 1 survival values ranged from 103 to 3 % relative to the solvent control. The test material did not cause a statistically significant increase the mutant frequency.

In the presence of S9 mix, in Test 1, cells were exposed to concentrations from5 to 400 µg/mL. As an insufficient toxicity range was achieved an additional test was performed. In this additional test, cells were exposed to concentrations from5 to 700 µg/mL. Day 1 survival values ranged from 108 to 21% relative to the solvent control. The test material did not cause a statistically significant increase the mutant frequency. Although the mutant frequency of the concurrent solvent control is outside of the stated accepted range (20.50 mutants per 106survivors) it is close to the historical maximum (20 mutants per 106survivors) and therefore was considered to be acceptable. Several of the test material test cultures also had mutation frequencies that exceeded the historical control maximum. However, there was no dose-response relationship or statistical significance so these data were considered to be acceptable and not to be indicative of a positive response.

In the absence of S9 mix, in Test 2, cells were exposed to concentrations from 5 to 100 µg/mL. Day 1 survival values ranged from 101 to 18% relative to the solvent control. The test material did not cause a statistically significant increase the mutant frequency. Several of the test material test cultures had mutation frequencies that exceeded the historical control maximum. However, there was no dose-response relationship or statistical significance so these data were considered to be acceptable and not to be indicative of a positive response.

In the presence of S9 mix, in Test 2, cells were exposed to concentrations from 5 to 700 µg/mL. Day 1 survival values ranged from 93 to 19% relative to the solvent control. The test material did not cause a statistically significant increase the mutant frequency. Several of the test material test cultures had mutation frequencies that exceeded the historical control maximum. However, there was no dose-response relationship or statistical significance so these data were considered to be acceptable and not to be indicative of a positive response.

 

The positive control treatments, both in the absence and presence of metabolic activation, induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation, in both experiments.

 

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1999-12-20 to 2000-05-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 473 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1992
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
according to Directive 88/320/EEC (Inspected on 1999-09-22)
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):
Human lymphocytes were prepared from whole blood samples obtained from two healthy donors (1 male and 1 female) and collected into heparinised sterile tubes.
- Type and identity of media: RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (l00 µg/mL) and phytohaemagglutinin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (15% S9-fraction obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg bw) by intraperitoneal route)
Test concentrations with justification for top dose:
- First experiment: up to 5000 µg/mL
- Second experiment: up to 2500 µg/mL
- Third experiment (-S9): up to 1250 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance not sufficiently water soluble
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment)
Positive control substance:
mitomycin C
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
50 µg/mL
Positive control substance:
cyclophosphamide
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: cells cultured 48 hours at 37°C
- Exposure duration: First experiment: 3 hours (+/-) S9-mix
Second experiment: 20 hours and 44 hours (-)S9-mix, 3 hours (+)S9-mix
Third experiement: 20 hours and 44 hours (-)S9-mix
- Expression time (cells in growth medium): First experiment: 17 hours
Second experiment: 17 hours and 41 hours (+)S9-mix
- Fixation time (start of exposure up to fixation or harvest of cells): First experiment 20 hours (+/-)S9-mix
Second experiment : 20 hours and 44 hours
Third experiment: 20 hours and 44 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (10 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level, with 100 metaphases/culture whenever possible

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Rationale for test conditions:
Tested up to cytotoxic concentrations.
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.
Statistics:
For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the X² test, in which p = 0.05 was used as the lowest level of significance.
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effect: at the highest dose-level, the pH was about 7.4 as for vehicle control.
- Effects of osmolality: No effect: at the highest dose-level, the osmolality equal to 366 mOsm/kg H2O (372 mOsm/kg H2O for the vehicle control).
- Evaporation from medium: Not expected due to low vapor pressure
- Water solubility: The test substance was not sufficiently soluble in water for water to be used as the vehicle.
- Precipitation: an important emulsion was observed at 5000 µg/mL and a slight to moderate emulsion at dose-levels equal or above 1250 µg/mL.

RANGE-FINDING/SCREENING STUDIES: not performed

COMPARISON WITH HISTORICAL CONTROL DATA: The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were consistent with acceptance criteria. The study was therefore considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Without S9-mix:
In the first experiment, the test substance was moderately to strongly toxic, mainly at dose-levels ~ 1250 µg/mL: a 49-100% decrease in the mitotic index was noted. In the second experiment, for the 20-hour treatment time, the test substance was completely toxic at dose-level ~ 1250 µg/mL. At the lower dose-levels, a slight toxicity was induced between 156.25 and 625 µg/mL. For the 44-hour treatment time, a slight to strong toxicity was observed as shown by a 7-100% decrease in the mitotic index. On the basis of International Guidelines and in order to reach a more adequate level of toxicity than that obtained in the second experiment (more than 50% decrease in the mitotic index), a third experiment was performed under the same experimental conditions, using a closer range of dose-levels. In the third experiment, for the 20-hour treatment time, the test substance was completely toxic at 1250 µg/mL. At the lower dose-levels, a 5-59% decrease in the mitotic index was noted but without clear evidence of a dose-relationship. At the 44-hour treatment time, a 18-95% decrease in the mitotic index was recorded.
- With S9-mix:
In the first experiment, a moderate to strong toxicity was induced at all dose-levels, as shown by a 33-93% decrease in the mitotic index. In the second experiment, a slight to strong toxicity was induced: 15-88% and 54-100% decreases in the mitotic index were noted at the 20-hour and 44-hour harvest time, respectively.

Table 7.6.1/1 Results of chromosome analysis (mean) – Experiment 1 without metabolic activation (3-hour treatment; 20-hour harvest)

 

Control

312.5 µg/mL

625 µg/mL

1250 µg/mL

Mitomycin C

Number of cells scored

200

200

200

200

100

Cytotoxicity

no

no

no

yes

yes

                                                   Mean

Gaps

0

1

0

0

3

Chromatid aberrations (/200)

breaks

3

4

2

3

73

exchanges

0

0

0

0

30

Isochromatid aberrations (/200)

breaks

0

0

2

3

10

exchanges

0

0

0

0

0

Frequency of aberrant cell (%)

+ Gaps

1.5

2.5

2.0

1.5

61.0

- Gaps

1.5

2.0

2.0

1.5

60.0*

Mitotic index(% of control)

100

81

98

33

28

Mitotic index

4.00

3.25

3.90

1.30

1.10

Polyploidy/Endoreplication

0

1

0

0

0

* = p < 0.001

 

Table 7.6.1/2 Results of chromosome analysis (mean) – Experiment 1 with metabolic activation (3-hour treatment, 20-hour harvest)

 

Control

312.5 µg/mL

625 µg/mL

1250 µg/mL

CPA

Number of cells scored

200

200

200

200

100

Cytotoxicity

no

no

no

yes

yes

                                                   Mean

Gaps

0

0

1

3

0

Chromatid aberrations (/200)

breaks

2

3

6

4

2

exchanges

0

0

0

0

78

Isochromatid aberrations (/200)

breaks

2

2

2

1

6

exchanges

2

0

0

0

2

Frequency of aberrant cell (%)

+ Gaps

3.0

2.0

4.0

3.5

52.0

- Gaps

3.0

2.0

3.5

2.5

52.0*

Mitotic index(% of control)

100

53

67

45

31

Mitotic index

3.75

2.00

2.50

1.70

1.15

Polyploidy/Endoreplication

1

0

0

0

0

* = p < 0.001

 

Table 7.6.1/3 Results of chromosome analysis (mean) – Experiment 2 without metabolic activation (20-hour treatment, 20-hour harvest)

 

Control

156.25 µg/mL

312.5 µg/mL

625 µg/mL

Mitomycin C

Number of cells scored

200

200

200

200

100

Cytotoxicity

no

no

no

yes

no

                                                   Mean

Gaps

0

1

2

1

2

Chromatid aberrations (/200)

breaks

2

2

4

3

25

exchanges

0

0

0

0

5

Isochromatid aberrations (/200)

breaks

1

0

1

0

2

exchanges

0

0

0

0

0

Frequency of aberrant cell (%)

+ Gaps

1.5

1.5

3.5

2.5

31.0

- Gaps

1.5

1.0

2.5

2.0

29.0*

Mitotic index(% of control)

100

85

84

0

88

Mitotic index

4.85

4.10

4.05

0.00

4.25

Polyploidy/Endoreplication

1

0

0

2

0

* = p < 0.001

 

Table 7.6.1/4 Results of chromosome analysis (mean) – Experiment 2 without metabolic activation (44-hour treatment, 44-hour harvest)

 

Control

156.25 µg/mL

Number of cells scored

200

200

Cytotoxicity

no

no

Mean

Gaps

0

0

Chromatid aberrations (/200)

breaks

1

1

exchanges

0

0

Isochromatid aberrations (/200)

breaks

0

0

exchanges

1

0

Frequency of aberrant cell (%)

+ Gaps

0.5

1

- Gaps

0.0

1

Mitotic index(% of control)

100

52

Mitotic index

2.80

1.45

Polyploidy/Endoreplication

0

2

* = p < 0.001

 

Table 7.6.1/5 Results of chromosome analysis (mean) – Experiment 2 with metabolic activation (3 -hour treatment, 20-hour harvest)

 

Control

312.5 µg/mL

625 µg/mL

1250 µg/mL

CPA

Number of cells scored

200

200

200

200

100

Cytotoxicity

no

no

yes

yes

yes

                                                   Mean

Gaps

2

1

2

3

3

Chromatid aberrations (/200)

breaks

2

3

6

2

87

exchanges

0

0

0

0

12

Isochromatid aberrations (/200)

breaks

0

1

1

0

0

exchanges

0

0

0

0

1

Frequency of aberrant cell (%)

+ Gaps

2.0

3.0

4.5

2.5

61.0

- Gaps

1.0

2.0

3.5

1.0

61.0*

Mitotic index(% of control)

100

85

49

32

16

Mitotic index

4.40

3.75

2.15

1.40

0.70

Polyploidy/Endoreplication

1

1

2

5

0

* = p < 0.001

 

Table 7.6.1/6 Results of chromosome analysis (mean) – Experiment 2 with metabolic activation (3-hour treatment, 44 -hour harvest)

 

Control

312.5 µg/mL

Number of cells scored

200

200

Cytotoxicity

no

yes

Mean

Gaps

0

1

Chromatid aberrations (/200)

breaks

3

3

exchanges

0

0

Isochromatid aberrations (/200)

breaks

2

6

exchanges

0

0

Frequency of aberrant cell (%)

+ Gaps

2.0

3.0

- Gaps

2.0

2.5

Mitotic index(% of control)

100

35

Mitotic index

2.30

0.80

Polyploidy/Endoreplication

0

0

* = p < 0.001

 

Table 7.6.1/7 Results of chromosome analysis (mean) – Experiment 3 without metabolic activation (20-hour treatment; 20-hour harvest)

 

Control

250 µg/mL

500 µg/mL

750 µg/mL

Mitomycin C

Number of cells scored

200

200

200

200

100

Cytotoxicity

no

no

no

no

no

                                                   Mean

Gaps

2

5

6

5

0

Chromatid aberrations (/200)

breaks

0

2

1

0

9

exchanges

0

0

0

0

32

Isochromatid aberrations (/200)

breaks

0

2

1

0

12

exchanges

0

1

0

0

3

Frequency of aberrant cell (%)

+ Gaps

0.5

4.0

4.0

2.5

41.0

- Gaps

0.0

2.0

1.0

0.0

37.0*

Mitotic index(% of control)

100

91

61

70

59

Mitotic index

2.20

2.00

1.35

1.55

1.30

Polyploidy/Endoreplication

0

2

3

1

0

* = p < 0.001

 

Table 7.6.1/8 Results of chromosome analysis (mean) – Experiment 3 without metabolic activation (44-hour treatment; 44-hour harvest)

 

Control

125 µg/mL

Number of cells scored

200

200

Cytotoxicity

no

no

Mean

Gaps

4

8

Chromatid aberrations (/200)

breaks

0

0

exchanges

0

0

Isochromatid aberrations (/200)

breaks

2

0

exchanges

0

0

Frequency of aberrant cell (%)

+ Gaps

3.0

4.0

- Gaps

1.0

0.0

Mitotic index(% of control)

100

65

Mitotic index

2.75

1.80

Polyploidy/Endoreplication

1

0

* = p < 0.001

 

Conclusions:
Under the test conditions, the test material was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to ST 32 C 99 diluted in DMSO.

 

No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used for the first experiment and dose-levels for scoring of chromosomal aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI). 

 

The following treatment conditions were used:

- In the first experiment: 3 hours exposure with or without the addition of an induced rat liver homogenate metabolising system (S9-mix) with cell harvest after 17-hour expression period. Up to 5000 µg/mL.

- In the second experiment: 20 and 44-hour continuous exposure without S9-mix, 3-hour exposure followed by 17 and 41-hour expression period with S9-mix. Up to 2500 µg/mL.

- In the third experiment: 20 and 44-hour continuous exposure without the addition of S9-mix. Up to 1250 µg/mL.

 

Without S9-mix, ST 32 C 99 demonstrated slight to very strong toxicity, depending on the dose-levels and the treatment time. On the basis of International Guidelines and in order to reach a more adequate level of toxicity than that obtained in the second experiment (more than 50% decrease in the mitotic index), a third experiment was performed under the same experimental conditions, using a closer range of dose-levels.

 

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times.

 

Under the test conditions, ST 32 C 99 was considered to be non-clastogenic to human lymphocytes in vitro.

 

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Safepharm, 1999

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537

TA 98

TA 100

WP2 uvrA-

-S9

+S9

Up to 5000 µg/plate

(limit concentration)

-S9 : non mutagenic

+S9 : non mutagenic

2

 

HLS, 2012

CHO/hprt test (OECD 476)

K, rel. 1

Gene mutation

Chinese Hamster ovary cells

-S9

+S9

-S9: Up to 140 µg/mL

+S9: Up to 700 µg/mL

(up to cytotoxic concentrations)

-S9 : non mutagenic

+S9 : non mutagenic

3

 

CIT, 2000

HL CAT

(OECD 473)

K, rel. 1

Chromosomal aberration

Human Lymphocytes

 

-S9

+S9

Up to 5000 µg/mL

(up to limit or cytotoxic concentrations)

-S9 : non clastogenic

+S9 : non clastogenic

 

Gene mutation Assays (Test n° 1-2):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

Inability to produce gene mutation was confirmed in mammals using an in vitro forward mutation assay in Chinese hamster ovary cells (CHO/hprt test)(Test n°2). None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. The substance does not induce forward mutations at the hprt locus in CHO cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. The substance is therefore considered as negative for inducing forward mutations at the hprt locus in CHO cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.

 

 

Chromosomal aberration (Test n°3)

The clastogenic potential of the substance was determined using an in vitro chromosome aberration test in human lymphocytes (OECD 473), which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of three experiments. The substance does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. The substance is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Based on the available data, no additional classification is proposed according to the CLP or the GHS.