Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene Mutation Study in Bacteria: Negative with and without metabolic activation in S. typhimurium TA98, TA100, TA1535 and TA1537 and E.coli WP2uvrA
- Gene Mutation Study in Mammalian Cells: Negative with and without metabolic activation in mouse lymphoma cells

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:
30 December 1988 to 13 October 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
The study protocol states that it was written to comply with the standardised guideline OECD 471.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Properly maintained: Yes. Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37 ± 2 °C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titre of approximately 10^9 cells per millilitre. The actual titres were determined by viable count assays on nutrient agar plates.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Properly maintained: Yes. Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37 ± 2 °C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titre of approximately 10^9 cells per millilitre. The actual titres were determined by viable count assays on nutrient agar plates.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
- Mutagenicity assay (Experiment B1): 25, 75, 200, 600, 1800 and 5000 µg/plate
Vehicle / solvent:
- Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Based on solubility of the test material, permitting preparation of a soluble or workable stock concentration up to 500 mg/L.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
On the day of its use, minimal top agar, containing 0.8 % agar (w/v) and 0.5 % NaCl (w/v), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 µM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. Water was sterile, deionised water produced by the Milli-Q Reagent Water System. Bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar and supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No. 2 (dry powder).
Test material dilutions were prepared immediately before use. 0.5 mL of S9 or Sham mix, 100 µL of tester strain and 50 µL of vehicle, test or positive control material were added to 2.0 mL of molten selective top agar at 45 ± 2 °C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar.

DURATION
- Exposure duration: After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37 ± 2 °C. Plates that were not counted immediately following the incubation period were stored at 2 to 8 °C until colony counting could be conducted.

NUMBER OF REPLICATIONS: Plated in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test material toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.

OTHER: Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
EVALUATION CRITERIA
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated. For the test material to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test material. Data sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA98, TA100 and WP2uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

CRITERIA FOR A VALID TEST
The following criteria must be met for the mutagenicity assay to be considered valid.
All Salmonella and E. coli tester strain cultures must demonstrate the appropriate characteristics.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
To ensure that appropriate numbers of bacteria are plated, tester strain culture titres must be greater than or equal to 0.3 x 10^9 cells/mL.
The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the respective vehicle control.
A minimum of three non-toxic dose levels are required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn.
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitate was observed but toxicity was generally observed at 5000 µg per plate. In Experiment B1, no positive responses were observed with any of the tester strains in the presence and absence of S9 activation. However, a non-dose responsive increase was observed with tester strain TA 1537 (1.7-fold, maximum increase) in the presence of S9 activation. This strain/activation condition was retested in Experiment B2 to clarify the response observed. In Experiment B2, no positive response was observed with tester strain TA1537 in the presence of S9 activation.
All criteria for a valid study were met. The results indicate that under the conditions of this study the test material did not cause a positive response with any of the tester strains in the presence and absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a full independent repeat assay because no unique metabolism requirements were known about the test material and because the equivocal response was retested to resolve the nature of the response.

Table 1: Summary of Experiment B1

± S9 Mix

Concentration

(µg/plate)

Mean Revertants / plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

25

75

200

600

1800

5000

105

125

112

106

102

39

0

9

8

10

13

12

8

0

14

16

13

10

11

10

10

15

17

17

16

22

23

3

3

4

5

5

8

3

0

+

Solvent

25

75

200

600

1800

5000

119

121

126

122

131

151

30

13

13

13

13

14

8

1

16

11

17

13

14

12

12

17

19

23

23

23

28

8

7

6

8

5

5

12

5

Positive Controls

-

Name

SA

SA

MMS

2 NF

9AA

Concentration (µg/plate)

1.0

1.0

1000

1.0

75

Mean no. colonies/plate

514

421

166

241

423

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

1.0

1.0

10

1.0

1.0

Mean no. colonies/plate

623

70

533

429

62

MMS = Methyl methanesulfonate

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

2NF = 2-Nitrofluorene

Table 2: Summary of Experiment B2

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate in strain TA1537

+

Solvent

25

75

200

600

1000

1800

5000

7

6

7

7

7

4

8

3

Positive Controls

+

Name

2AA

Concentration (µg/plate)

1

Mean no. colonies/plate

62

2AA = 2-aminoanthracene

Conclusions:
Under the conditions of this study, the test material was concluded to be negative with regard to genotoxicity in the Bacterial Gene Mutation Assay.
Executive summary:

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted using a protocol written to comply with the standardised guideline OECD 471 under GLP conditions.

S. typhimurium tester strains TA98, TA100, TA1535 and TA1537 and E.coli tester strain WP2uvrA were exposed to the test material in the presence and absence of Aroclor-induced rat liver S9 using the plate incorporation method. Following a preliminary toxicity assay, the mutagenicity assay (Experiment B1) was used to evaluate the mutagenic potential of the test material at concentrations of 0, 25, 75, 200, 600, 1800 and 5000 µg/plate in DMSO. Appropriate vehicle and positive controls for each tester strain were evaluated concurrently. All dose levels of test material, vehicle controls and positive controls were plated in triplicate.

No precipitate was observed but toxicity was generally observed at 5000 µg per plate. In Experiment B1, no positive responses were observed with any of the tester strains in the presence and absence of S9 activation. However, a non-dose responsive increase was observed with tester strain TA 1537 (1.7-fold, maximum increase) in the presence of S9 activation. This strain/activation condition was retested in Experiment B2 to clarify the response observed. In Experiment B2, no positive response was observed with tester strain TA1537 in the presence of S9 activation.

All criteria for a valid study were met. The results indicate that under the conditions of this study the test material did not cause a positive response with any of the tester strains in the presence and absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a full independent repeat assay because no unique metabolism requirements were known about the test material and because the equivocal response was retested to resolve the nature of the response.

Under the conditions of this study, the test material was concluded to be negative with regard to genotoxicity in the Bacterial Gene Mutation Assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 September 2012 to 18 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
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
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
(METI/MHLW guidelines for testing of new chemical substances)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase TK +/- locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10 % donor horse serum (giving R10 media).
- Properly maintained: Yes. Stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were cultured in medium at 37 °C with 5 % CO2 in air.
- Periodically checked for Mycoplasma contamination: Yes. Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically "cleansed" against high spontaneous background: Yes. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2 % S9
Test concentrations with justification for top dose:
- Preliminary toxicity test: 7.51, 15.02, 30.03, 60.06, 120.13, 240.25, 480.5, 961 and 1922 μg/mL
- Experiment 1 with and without S9 mix (4 hour exposure): 7.5, 15, 30, 60, 120, 160, 200 and 240 μg/mL
- Experiment 2 without S9 mix (24 hour exposure): 0.94, 1.88, 3.75, 7.5, 15, 20, 30 and 60 μg/mL
- Experiment 2 with S9 mix (4 hour exposure): 30, 60, 120, 160, 200, 240, 280 and 320 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Selected following solubility checks performed in-house.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
Controls for test 1 and 2 conducted in the absence of metabolic activation.
Details on test system and experimental conditions:
TREATMENT OF THE CELLS
Experiment 1
1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universal vessels were utilised. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test material and for the vehicle and positive controls. To each universal vessel was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions and sufficient R0 medium to bring the total volume to 20 mL.
The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
1 x 10^6 cells/mL in 10 mL cultures in R10 medium were used for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm² tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test material and vehicle and positive controls. To each universal vessel was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions and sufficient R0 medium to give a final volume of 20 mL (R10 is used for the 24-hour exposure group).
The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then re-suspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and sub cultured every 24 hours for the expression period of two days by counting and diluting to 2 x 10^5 cells/mL.
On Day 2 of the experiment the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

PLATE SCORING AND DETERMINATION OF MUTANT COLONIES
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days' incubation at 37 °C with 5 % CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately 1/4 to 3/4 of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25 % of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies, 0.025 mL of Thiazolyl Blue Tetrazolium Bromide (MTT) solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

DETERMINATION OF CYTOTOXICITY
- Calculation of Percentage Relative Suspension Growth (%RSG)
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
Suspension Growth (SG) = (24-hour cell count / 2) x (48-hour cell count / 2)
Day 0 Factor = dose 0-hour cell count / vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor) / vehicle control SG] x 100

- Calculation of Day 2 Viability (%V)
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = number of negative wells / total wells plated
%V = (-lnP(0) x 100) / number of cells/well

- Calculation of Relative Total Growth (RTG)
For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = (%V / Mean Solvent Control %V) x 100 %

Finally, for each culture RTG is calculated:
RTG = (RCE x RSG) / 100 %

CALCULATION OF MUTATION FREQUENCY (MF)
MF per survivor = [-lnP(0) selective medium / cells per well in selective medium] / surviving fraction in non-selective medium
Evaluation criteria:
INTERPRETATION OF RESULTS
The normal range for mutant frequency per survivor is 50-170 x 10^-6 for the TK+/- locus in L5178Y cells at the testing laboratory. Vehicle controls results should ideally be within this range.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.
Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the Global Evaluation Factor (GEF) of 126 x 10^-6 for the microwell method and demonstrates a positive linear trend will be considered positive.
However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.
Statistics:
The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).
Dose levels that have RTG survival values less than 10 % are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The test material was shown to be more toxic in the 24 hour exposure group. The nature of the toxicity curve was very steep particularly in both 4-hour exposure groups. A precipitate of the test material was observed at and above 961 µg/mL in all three exposure groups. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test material-induced toxicity and dose levels above 320 µg/mL were not investigated.

EXPERIMENT 1
There was once again evidence of marked dose-related toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values. There was evidence of a marked reduction in viability (%V) at one dose level (200 µg/mL) in the absence of metabolic activation, however this dose level was excluded due to excessive toxicity. No marked reductions in viability (%V) were observed at any other dose level in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had not been achieved in either the absence or presence of metabolic activation. Optimum levels of toxicity were not achieved due the excessive toxicity at and above 200 µg/mL in the absence of metabolic activation and adequate toxicity was not achieved in the presence of metabolic activation. The toxicity observed in Experiment 1 was slightly less than that observed in the preliminary toxicity test and it was considered that it would be hard to achieve optimum toxicity due the fluctuating nature of the test material induced toxicity. The excessive toxicity observed at 240 µg/mL in the absence of metabolic activation resulted in this dose level not being plated for viability or 5-TFT resistance. In the absence of metabolic activation a dose level (200 µg/mL) that exceeded the upper acceptable limit of toxicity as indicated by the %RSG was plated out as there was sufficient cell culture available. This dose level was later excluded from statistical analysis as the RTG value also fell below the acceptable level of toxicity. Acceptable levels of toxicity were seen with both positive control substances.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test material did not induce any statistically significant or dose related (linear-trend) increase in the mutant frequency x 10^-6 per viable cell in the absence or presence of metabolic activation. No increase in mutant frequency above the GEF value was observed at 200 µg/mL in the absence of S9 which was excessively toxic. This was taken to indicate no mutagenic activity was occurring. No precipitate of the test material was observed at any dose level.

EXPERIMENT 2
As was seen previously, there was evidence of marked toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values. On this occasion there was evidence of a modest reduction in viability (%V) in the presence of metabolic activation, therefore indicating that residual toxicity had occurred in this exposure group. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at and above 30 µg/mL in the absence of metabolic activation and at 320 µg/mL in the presence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.
The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test material.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test material did induce a small but statistically significant dose related (linear-trend) increase in the mutant frequency x 10^-6 per viable cell in the presence of metabolic activation. However all of the mutant frequency values were within the acceptable range for a vehicle control and the GEF value was not exceeded therefore this result was considered to be of no toxicological significance. No precipitate of the test material was observed at any dose level in either in the absence or presence of metabolic activation.

Table 1: Summary of Results for Experiment 1

Dose (µg/mL)

4 Hour Treatment Without S9

Dose (µg/mL)

4 Hour Treatment With S9

%RSG

RTG

MF

%RSG

RTG

MF

0

100

1.00

119.57

0

100

1.00

101.68

7.5 Ø

90

 

 

7.5 Ø

82

 

 

15

88

0.85

119.76

15 Ø

82

 

 

30

82

0.74

111.51

30

77

0.86

113.25

60

72

0.65

127.02

60

77

0.86

100.51

120

49

0.46

136.40

120

70

0.76

99.01

160

43

0.40

141.99

160

68

0.66

101.31

200 X

2

0.01

203.14

200

67

0.64

95.43

240 Ø

1

 

 

240

57

0.53

140.40

EMS 400

78

0.66

877.98

CP 2

53

0.30

1702.71

%RSG = Relative Suspension Growth

RTG =   Relative Total Growth

MF§ =  5-TFT resistant mutants/10^6 viable cells 2 days after treatment

Ø = Not plated for viability or 5-TFT resistance

X = Treatment excluded from test statistics due to toxicity

EMS =  Ethylmethanesulphonate

CP = Cyclophosphamide

 

Table 2: Summary of Results for Experiment 2

Dose (µg/mL)

24 Hour Treatment Without S9

Dose (µg/mL)

4 Hour Treatment With S9

%RSG

RTG

MF

%RSG

RTG

MF

0

100

1.00

130.27

0

100

1.00

112.62

0.94

88

1.00

121.99

30 Ø

86

 

 

1.88

80

0.91

132.84

60

85

0.81

119.34

3.75

76

0.77

133.17

120

71

0.65

133.35

7.5

54

0.68

134.50

160

66

0.60

137.56

15

26

0.39

142.02

200

55

0.55

123.87

20

11

0.22

132.29

240

40

0.34

164.31

30 Ø

6

 

 

280

20

0.17

148.69

60 Ø

0

 

 

320 Ø

3

 

 

EMS 150

40

0.38

1021.69

CP 2

65

0.39

1161.31

%RSG = Relative Suspension Growth

RTG =   Relative Total Growth

MF§ =  5-TFT resistant mutants/10^6 viable cells 2 days after treatment

Ø = Not plated for viability or 5-TFT resistance

X = Treatment excluded from test statistics due to toxicity

EMS =  Ethylmethanesulphonate

CP = Cyclophosphamide

Conclusions:
Under the conditions of this study the test material is not mutagenic in the presence and absence of metabolic activation.
Executive summary:

The mutagenic activity of the test material was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The study was conducted in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300 under GLP conditions.

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

The dose range of test material was selected following the results of a preliminary toxicity test and was 7.5 to 240 µg/mL in the absence and presence of metabolic activation in Experiment 1. In Experiment 2 the dose range was 0.94 to 60 µg/mL in the absence of metabolic activation, and 30 to 320 µg/mL in the presence of metabolic activation.

The maximum dose levels used in the mutagenicity test were limited by test material-induced toxicity. No precipitate of the test material was observed at any dose level. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Under the conditions of this study the test material is not mutagenic in the presence and absence of metabolic activation.

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

Genetic toxicity in vivo

Description of key information

- Mammalian Erythroctye Micronucleus Test: Negative using male and female mice

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 December 1999 to 15 February 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
The study protocol states that it was written to comply with the standardised guideline OECD 474.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 to 8 weeks old
- Weights at study initiation: Weights at randomisation were within the following ranges: Males 27.5 - 31.5 g and females 25.2 - 27.5 g (pilot toxicity study); males 26.2 - 33.5 g and females 25.6 - 29.5 g (toxicity study); males 30.3 - 34.5 g and females 26.2 - 28.7 g (supplemental toxicity study); and males 29.1 - 33.5 g and females 25.4 - 30.0 g (micronucleus study)
- Assigned to test groups randomly: Yes. In the micronucleus assay, mice were assigned to experimental groups according to a computer-generated program which is based on distribution according to body weight.
- Fasting period before study: No
- Housing: Housed in an AAALAC-accredited facility with a controlled environment. Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and which were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet: ad libitum certified laboratory rodent chow
- Water: ad libitum tap water
- Acclimation period: no less than 5 days of quarantine

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 °F
- Humidity: 50 ± 20 % relative humidity
- Photoperiod: 12 hour light/dark cycle

IN-LIFE DATES
From: 21 December 1999 (pilot study); 04 January 2000 (toxicity study); 18 January 2000 (supplemental toxicity study); and 25 January 2000 (micronucleus study)
To: No data
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: Determined to be the solvent of choice based on information provided by the Sponsor and compatibility of the vehicle with the test system animals. The test material was soluble in corn oil at 100 mg/mL.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material, vehicle and positive control were administered at a constant volume of 20 mL/kg body weight.
Duration of treatment / exposure:
- Pilot study, toxicity assay and the supplemental toxicity study: 3 days
- Micronucleus study: 24 and 48 hours
Frequency of treatment:
Dosed once through a single intraperitoneal injection
Post exposure period:
In the pilot toxicity study, toxicity assay and the supplemental toxicity study, mice were observed after dose administration and daily thereafter for 3 days for clinical signs of chemical effect. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration.
For the micronucleus assay, bone marrow was collected from 5 animals per sex per dose group for all dose levels after 24 hours post dose and from 5 animals per sex from the vehicle control and high dose groups after 48 hours post dose.
Remarks:
Doses / Concentrations:
Pilot toxicity study: male mice were dosed with 1, 10, 100 or 1000 mg test material/kg body weight and male and female mice were dosed with 2000 mg/kg.
Basis:
other: actual administered
Remarks:
Doses / Concentrations:
Toxicity assay: male and female mice were dosed with 1200, 1400, 1600 or 1800 mg test material/kg body weight.
Basis:
other: actual administered
Remarks:
Doses / Concentrations:
Supplemental toxicity study: male and female mice were dosed with 1000 mg test material/kg body weight.
Basis:
other: actual administered
Remarks:
Doses / Concentrations:
Micronucleus assay: male and female mice were dosed with 181, 362 or 725 mg test material/kg body weight.
Basis:
other: actual administered
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide dissolved in sterile distilled water at a concentration of 2.5 mg/mL.
- Route of administration: Administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection.
- Doses / concentrations: 50 mg/kg
Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION
The LD50/3 in the supplemental toxicity study was calculated by probit analysis to be approximately 1034.5 mg/kg for male and female mice. The high dose for the micronucleus test was set at 725 mg/kg for male and female mice which was estimated to beapproximately70 % of the LD50/3.

DETAILS OF SLIDE PREPARATION
At the scheduled sacrifice times, five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL foetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS - SCORING FOR MICRONUCLEI
Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
EVALUATION OF TEST RESULTS
The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. All analyses were performed separately for each sex and sampling time.
In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
All conclusions were based on sound scientific judgement; however, as a guide to interpretation of the data, the test material was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p≤0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test material was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

CRITERIA FOR A VALID TEST
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5 %) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p≤0.05, Kastenbaum-Bowman Tables).
Statistics:
Statistical significance of the incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PILOT STUDY
Mortality occurred after dose administration in 5/5 males and 5/5 females at 2000 mg/kg. Clinical signs which were noted following dose administration included lethargy, piloerection, irregular breathing and crusty eyes in male mice at 1000 mg/kg and convulsions in males and females at 2000 mg/kg. All other animals appeared normal throughout the observation period.

TOXICITY ASSAY
Mortality occurred within two days of dose administration in 4/5 males and 5/5 females at 1200 mg/kg and all males and females at 1400, 1600 and 1800 mg/kg. Clinical signs which were noted after dose administration included convulsions in all animals at all dose levels and prostration, irregular breathing and crusty eyes in males and females at 1200, 1400, 1600 and 1800 mg/kg. Lethargy and piloerection were observed in one surviving male mouse at 1200 mg/kg.

SUPPLEMENTAL TOXICITY STUDY
Mortality was observed in 2/5 female mice. Clinical signs following dose administration included prostration, irregular breathing, crusty eyes, tremors, lethargy and piloerection in males and females at 1000 mg/kg.

MICRONUCLEUS ASSAY
One female mouse receiving 725 mg/kg was found dead on the day following dose administration and was replaced at the time of bone marrow collection with an animal from a replacement group which had also been dosed with 725 mg/kg. Mortality in the other test material-treated groups was not observed. Clinical signs which were noted after dose administration included lethargy and piloerection in males and females at 362 and 725 mg/kg and prostration and irregular breathing in animals at 725 mg/kg. All other mice treated with the test and control materials appeared normal during the study.
Slight reductions of 3 to 12 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test material-treated groups relative to their respective vehicle controls. Reductions were observed in female dose groups 24 hours after treatment with 181, 362 and 725 mg/kg and in the male dose group 48 hours after treatment with 725 mg/kg. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test material-treated groups was not statistically increased relative to their respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time (p>0.05, Kastenbaum-Bowman Tables). Cyclophosphamide (positive control) induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p≤0.05, Kastenbaum-Bowman Tables).

Table 1: Summary of Bone Marrow Micronucleus Study

Treatment (mg/kg)

Sex

Time (hours)

No. of Mice

PCE/Total Erythrocytes (Mean ± SD)

Change from Control (%)

Micronucleated Polychromatic Erythrocytes

No./1000 PCEs (Mean ± SD)

No./PCEs Scored

Vehicle

M

24

5

0.483 ± 0.04

-

0.3 ± 0.27

3/10 000

F

5

0.568 ± 0.03

-

0.9 ± 0.55

9/10 000

181

M

5

0.487 ± 0.11

1

0.1 ± 0.22

1/10 000

F

5

0.549 ± 0.04

-3

0.6 ± 0.22

6/10 000

362

M

5

0.535 ± 0.04

11

0.5 ± 0.00

5/10 000

F

5

0.529 ± 0.04

-7

0.4 ± 0.42

4/10 000

725

M

5

0.546 ± 0.06

13

0.3 ± 0.27

3/10 000

F

5

0.536 ± 0.05

-6

0.5 ± 0.35

5/10 000

CP 50

M

5

0.571 ± 0.05

18

25.6 ± 5.24

256/10 000*

F

5

0.549 ± 0.09

-3

18.3 ± 6.47

183/10 000*

Vehicle

M

48

5

0.615 ± 0.04

-

0.2 ± 0.27

2/10 000

F

5

0.553 ± 0.03

-

0.3 ± 0.45

3/10 000

725

M

5

0.540 ± 0.07

-12

0.0 ± 0.00

0/10 000

F

5

0.554 ± 0.04

0

0.5 ± 0.50

5/10 000

*p≤0.05 (Kastenbaum-Bowman Tables)

Conclusions:
Under the conditions of the study, the test material was concluded to be negative in the micronucleus test using male and female mice.
Executive summary:

A mammalian erythrocyte micronucleus test in ICR mice was conducted to assess the toxicity of the test material using a protocol written to comply with the standardised guideline OECD 474 under GLP conditions.

The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot toxicity study followed by a toxicity study and supplemental toxicity study. The second phase, the micronucleus study, evaluated the potential of the test material to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control materials were administered in a constant volume of 20 mL/kg body weight through single intraperitoneal injection. Corn oil was determined to be the solvent of choice based on compatibility of the vehicle with the test system animals..

In the pilot toxicity study, male mice were dosed with 1, 10, 100 or 1000 mg test material/kg body weight and male and female mice were dosed with 2000 mg/kg (5 animals per sex per dose level). Mortality was observed in 5/5 male mice and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included lethargy, piloerection, crusty eyes and irregular breathing in all males at 1000 mg/kg and convulsions in all males and females at 2000 mg/kg.

In the toxicity assay, male and female mice were dosed with 1200, 1400, 1600 or 1800 mg test material/kg body weight (5 animals per sex per dose level). Mortality was observed in 4/5 male mice and 5/5 female mice at 1200 mg/kg and in all males and females at 1400, 1600 and 1800 mg/kg. Clinical signs following dose administration included convulsions in all animals at all dose levels and prostration, irregular breathing and crusty eyes in males and females at 1200, 1400, 1600 and 1800 mg/kg. Lethargy and piloerection were observed in one surviving animal at 1200 mg/kg. Due to high mortality at 1200 mg/kg and clinical signs at 1000 mg/kg (pilot study), a supplemental toxicity study was performed using the test material at a dose level of 1000 mg/kg.

In the supplemental toxicity study, male and female mice were dosed with 1000 mg test material/kg body weight (5 animals per sex per dose level). Mortality was observed in 2/5 female mice. Clinical signs following dose administration included prostration, irregular breathing, crusty eyes, tremors, lethargy and piloerection in males and females at 1000 mg/kg. The high dose for the micronucleus test was set at 725 mg/kg which was estimated to be approximately 70 % of the LD50/3.

In the micronucleus assay, male and female mice were dosed with 0, 181, 362 or 725 mg test material/kg body weight (15 animals per sex per dose). A concurrent positive control group was dosed with cyclophosphamide. Mortality was observed in 1/15 female mice receiving 725 mg/kg. Clinical signs following dose administration included lethargy and piloerection in male and female mice at 362 and 725 mg/kg and prostration and irregular breathing in males and females at 725 mg/kg. Bone marrow cells, collected 24 and 48 hours after treatment (5 animals per sex per dose per time interval), were examined microscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 12 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test material-treated groups relative to the respective vehicle controls. These reductions suggest that the test material did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test material-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration.

CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice.

Under the conditions of the study, the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female mice.

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

Additional information

In vitro data

- Bacterial reverse mutation

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted using a protocol written to comply with the standardised guideline OECD 471 under GLP conditions. The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

S. typhimurium tester strains TA98, TA100, TA1535 and TA1537 and E.coli tester strain WP2uvrA were exposed to the test material in the presence and absence of Aroclor-induced rat liver S9 using the plate incorporation method. Following a preliminary toxicity assay, the mutagenicity assay (Experiment B1) was used to evaluate the mutagenic potential of the test material at concentrations of 0, 25, 75, 200, 600, 1800 and 5000 µg/plate in DMSO. Appropriate vehicle and positive controls for each tester strain were evaluated concurrently. All dose levels of test material, vehicle controls and positive controls were plated in triplicate.

No precipitate was observed but toxicity was generally observed at 5000 µg per plate. In Experiment B1, no positive responses were observed with any of the tester strains in the presence and absence of S9 activation. However, a non-dose responsive increase was observed with tester strain TA 1537 (1.7-fold, maximum increase) in the presence of S9 activation. This strain/activation condition was retested in Experiment B2 to clarify the response observed. In Experiment B2, no positive response was observed with tester strain TA1537 in the presence of S9 activation.

All criteria for a valid study were met. The results indicate that under the conditions of this study the test material did not cause a positive response with any of the tester strains in the presence and absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a full independent repeat assay because no unique metabolism requirements were known about the test material and because the equivocal response was retested to resolve the nature of the response.

Under the conditions of this study, the test material was concluded to be negative with regard to genotoxicity in the Bacterial Gene Mutation Assay.

- Gene mutation in mammalian cells

The mutagenic activity of the test material was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The study was conducted in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

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

The dose range of test material was selected following the results of a preliminary toxicity test and was 7.5 to 240 µg/mL in the absence and presence of metabolic activation in Experiment 1. In Experiment 2 the dose range was 0.94 to 60 µg/mL in the absence of metabolic activation, and 30 to 320 µg/mL in the presence of metabolic activation.

The maximum dose levels used in the mutagenicity test were limited by test material-induced toxicity. No precipitate of the test material was observed at any dose level. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Under the conditions of this study the test material is not mutagenic in the presence and absence of metabolic activation.

In vivo data

- Chromosome aberration

A mammalian erythrocyte micronucleus test in ICR mice was conducted to assess the toxicity of the test material using a protocol written to comply with the standardised guideline OECD 474 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot toxicity study followed by a toxicity study and supplemental toxicity study. The second phase, the micronucleus study, evaluated the potential of the test material to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control materials were administered in a constant volume of 20 mL/kg body weight through single intraperitoneal injection. Corn oil was determined to be the solvent of choice based on compatibility of the vehicle with the test system animals..

In the pilot toxicity study, male mice were dosed with 1, 10, 100 or 1000 mg test material/kg body weight and male and female mice were dosed with 2000 mg/kg (5 animals per sex per dose level). Mortality was observed in 5/5 male mice and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included lethargy, piloerection, crusty eyes and irregular breathing in all males at 1000 mg/kg and convulsions in all males and females at 2000 mg/kg.

In the toxicity assay, male and female mice were dosed with 1200, 1400, 1600 or 1800 mg test material/kg body weight (5 animals per sex per dose level). Mortality was observed in 4/5 male mice and 5/5 female mice at 1200 mg/kg and in all males and females at 1400, 1600 and 1800 mg/kg. Clinical signs following dose administration included convulsions in all animals at all dose levels and prostration, irregular breathing and crusty eyes in males and females at 1200, 1400, 1600 and 1800 mg/kg. Lethargy and piloerection were observed in one surviving animal at 1200 mg/kg. Due to high mortality at 1200 mg/kg and clinical signs at 1000 mg/kg (pilot study), a supplemental toxicity study was performed using the test material at a dose level of 1000 mg/kg.

In the supplemental toxicity study, male and female mice were dosed with 1000 mg test material/kg body weight (5 animals per sex per dose level). Mortality was observed in 2/5 female mice. Clinical signs following dose administration included prostration, irregular breathing, crusty eyes, tremors, lethargy and piloerection in males and females at 1000 mg/kg. The high dose for the micronucleus test was set at 725 mg/kg which was estimated to be approximately 70 % of the LD50/3.

In the micronucleus assay, male and female mice were dosed with 0, 181, 362 or 725 mg test material/kg body weight (15 animals per sex per dose). A concurrent positive control group was dosed with cyclophosphamide. Mortality was observed in 1/15 female mice receiving 725 mg/kg. Clinical signs following dose administration included lethargy and piloerection in male and female mice at 362 and 725 mg/kg and prostration and irregular breathing in males and females at 725 mg/kg. Bone marrow cells, collected 24 and 48 hours after treatment (5 animals per sex per dose per time interval), were examined microscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 12 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test material-treated groups relative to the respective vehicle controls. These reductions suggest that the test material did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test material-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration.

CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice.

Under the conditions of the study, the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female mice.


Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available genetic toxicity studies.