Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria: not mutagenic
- OECD 471; GLP; S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvrA; with and without S9; 20-5000 µg/plate; TA 1535 with S9 only, weakly non-dose dependent response, but never reaching the factor of three, which is required for a postive result. TA 1535 without S9 and TA 1537, TA 98, TA 100, E.coli WP2 uvrA with and without S9: negative

(2003).

- OECD 471; GLP not specified; S. typhimurium TA 1535, TA 1537, TA 98, TA 100; with and without S9; 20-5000 µg/plate; not mutagenic (1989)

- Equivalent to OECD 471; GLP not specified; S. typh. TA 1535, TA 1537, TA 98, TA 100; with and without S9; 0.005-50.0 µl per plate; not mutagenic (1980)


Gene mutation in mammalian cells:

- OECD 476; GLP; CHO cells; with and without S9; 0.23-140 µg/ml; not mutagenic (2015)

- Non-guideline; non-GLP; mouse lymphoma L5178Y; with and without S9; 0.0977-75 nl/ml; induced toxicity-related increases in mutant frequency - but only at highyl toxic concentrations (relativ growth -S9/+S 13/30 %) (1980)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
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)
Version / remarks:
adopted 1997
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Physical state: liquid
- Analytical purity: 86.3%
- Purity test date: 2014-07-30
- Lot/batch No.: 140023P040
- Expiration date of the lot/batch: 2015-07-25
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: stability was verified at room temperature in DMSO for at least 4h.
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 with or without (only during treatment with S9) FCS. All media were supported with 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
First experiment:
Without S9: 0.23, 0.47, 0.94, 1.88, 3.75, 7.5, 15, 30µg/mL
With S9: 1.88, 3.75, 7.5, 15, 30, 60, 120µg/mL

2nd experiment
Without S9: 0.27, 0.55, 1.09, 2.19, 4.38, 8.75, 17.5µg/mL
With S9: 2.19, 4.38, 8.75, 17.5, 35, 70, 140µg/mL

Third experiment
Without S9: 0.27, 0.55, 1.09, 2.19, 4.38, 8.75, 17.5µg/mL

In all experiments, at least four concentrations were evaluated.
Vehicle / solvent:
DMSO
Due to the insolubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 4h
- Expression time (cells in growth medium): 6-8 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the vehicle controls should be within our historical negative control data range of 0.00 – 16.43 mutants per 106 clonable cells
• The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies (historical positive control data
• At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a doseresponse relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.
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: with S9 mix, the pH was lowered at concentrations at and above 3012.5µg/mL
- Precipitation: precipitation in the culture medium (but not in the vehicle) occured at concentrations of 1506.3µg/mL and above (with or without S9 mix)
These concentrations are well above those used in the study due to high cytotoxicity. No influence on the result is expected.

RANGE-FINDING/SCREENING STUDIES: The doses were selected to reduce cloning efficiency to about 20% or less.

COMPARISON WITH HISTORICAL CONTROL DATA:
The positive control (EMS) without S9 mix did not produce the expected increase in mutant colonies. That's why this experiment was repeated - designated experiment 3. All other positive and negative control values were within the historical range. Also, mutant frequencies of the treatment groups did not exceed the historical negative control data.
Experimenta result without S9
Experiment Test group Mutant frequency [per 106cells] Cytotoxicity CE1 [%] Cytotoxicity CE2 [%]
1 vehicle control 0.61 100.0 100.0
0.23 n.c. 97.7 n.c.
0.47 n.c. 102.4 n.c.
0.94 0.32 96.9 95.6
1.88 0.63 96.8 95.9
3.75 5.20 90.7 91.1
7.50 1.73 49.9 65.5
15.00 n.c. 0.1 n.c.
30.00 n.c. 0.0 n.c.
positive control 111.10 96.8 96.0
2 vehicle control 5.72 100.0 100.0
0.27 n.c. 95.3 n.c.
0.55 6.06 96.9 116.5
1.09 1.55 86.5 105.1
2.19 1.99 84.1 109.1
4.38 0.35 87.9 96.2
8.75 n.c. 1.1 n.c.
17.50 n.c. 0.3 n.c.
positive control 5.18 88.5 119.8
Positive control did not fulfill acceptance criteria
3 vehicle control 6.41 100.0 100.0
0.27 n.c. 86.5 n.c.
0.55 n.c. 97.2 n.c.
1.09 3.93 86.2 85.8
2.19 5.29 79.9 83.2
4.38 1.78 77.5 86.6
8.75 0.00 35.8 74.2
17.50 n.c. 0.0 n.c.
positive control 111.19 83.9 73.2

Experimenta result with S9
Experiment Test group Mutant frequency [per 106cells] Cytotoxicity CE1 [%] Cytotoxicity CE2 [%]
1 vehicle control 1.71 100.0 100.0
1.88 n.c. 102.5 n.c.
3.75 n.c. 99.6 n.c.
7.50 4.00 98.2 93.1
15.00 2.65 94.4 102.4
30.00 10.43 66.7 95.1
60.00 2.07 30.6 102.7
120.00 n.c. 0.0 n.c.
positive control 120.67 101.6 102.0
2 vehicle control 1.73 100.0 100.0
2.19 n.c. 94.0 n.c.
4.38 1.35 83.6 102.4
8.75 1.00 50.8 100.5
17.50 3.55 38.7 100.2
35.00 7.01 24.1 93.3
70.00 n.c. 0.2 n.c.
140.00 n.c. 0.2 n.c.
positive control 89.81 107.1 93.0

Vehicle controls (culture medium, DMSO, acetone, ethanol, tetrahydrofurane)

Without S9 With S9
Mutant frequency uncorrected corrected* uncorrected corrected*
Mean 2,51 3,09 2,29 2,84
Min 0,00 0,00 0,00 0,00
Max 13,33 16,43 14,44 16,12
SD 2,57 3,18 2,35 2,94
Number of experiments 131 239

* = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period

Conclusions:
Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Mouse Lymphoma Forward Mutation Assay according to Clive D, Spector JFS (1975): Mutat. Res. 31, 17-29
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): C-150
- Physical state: liquid
- Analytical purity: no data
no further data
Target gene:
thymidine kinase (TK) gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's mouse leukemia medium supplemented with L-glutamine, sodium pyruvate and horse serum (10% by volume).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: heterozygous at the TK locus (TK +/-)
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
1st experiment without S-9 mix: 0.0977, 1.56, 3.13, 6.25 and 12.5 nl/ml; with S-9 mix: 3.12, 6.25, 12.5, 25 and 50 nl/ml
2nd experiment without S-9 mix: 2, 6, 8, 10 (twice) nl/ml; with S-9 mix: 2.5, 20, 40, 50 75 nl/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: the test substance was immiscible in water at 100 µl/ml but dissolved easily in DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate (EMS) and dimethylnotrosamine (DMN)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): after 10 days


SELECTION AGENT (mutation assays): 5-trifluorothymidine or 5-bromo-2'-deoxyuridine


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The following test results must be obtained:
1. A dose-related or toxicity-related increase in mutant frequency should be observed. It is desirable to obtain this relation for at least three doses, but this depends on the concentration steps chosen for the assay and the toxicity at which mutagenic activity appears.
2. If an increase of about two times the minimum criterion (150% of the concurrent background frequency plus 10x10 to the power of -6) or greater is observed for a single dose near the highest testable toxicity, the test material will be considered mutagenic. Smaller increases at a single dose near the highest testable toxicity will require confirmation by a repeat assay.
3. For some test materials, the correlation between toxicity and applied concentration is poor. The proportion of the applied material that effectively
interacts with the cells to cause genetic alterations is not always repeatable or under control. Conversely, measurable changes in frequency of inducted mutants may occur with concentration changes that cause only small changes in observable toxicity. Therefore, either parameter, applied
concentration or toxicity (percent relative growth), can be used to establish whether the mutagenic activity is related to an increase in effective
treatment. A negative correlation with dose is acceptable only if a positive correlation with toxicity exists. An apparent increase in mutagenic activity as a function of decreasing toxicity is not acceptable evidence for mutagenicity.
4. Treatments that induce less than 10% relative growth are included in the assay, but are not used as primary evidence for mutagenicity as it relates to risk assessment.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1.56 nl/ml (- S9); 40 nl/ml (+ S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Summary of results

Experiment 1:

without metabolic activation

concentration (nl/ml)

relative total growth (%)

mutant frequency

solvent control

100

24.7

positive control (5 µl/ml)

10.7

795.6

0.0977

25.5

36.3

1.56

11.6

28.3

3.13

13.9

39.6

6.25

8.9

42.7

12.5

0.9

95.8

 

with metabolic activation

concentration (nl/ml)

relative total growth (%)

mutant frequency

solvent control

100

30.5

positive control (DMN 3 µl/ml)

12.1

265.8

3.13

59.6

46.9

6.25

53.4

46.0

12.5

51.3

44.9

25

30.5

72.2

50

11.2

89.7

Experiment 2:

without metabolic activation

concentration (nl/ml)

relative total growth (%)

mutant frequency

solvent control

100

12.8

positive control (5 µl/ml)

23.2

425.7

2

35.2

18.5

6

16.2

28.4

8

23.0

30.3

10

9.5

55.9

10

10.7

41.6

 

with metabolic activation

concentration (nl/ml)

relative total growth (%)

mutant frequency

solvent control

100

18.2

positive control (DMN 3 µl/ml)

15.3

241.2

2.5

99.9

34.9

20

50.2

39.7

40

27.1

38.9

50

22.3

63.4

75

15.7

75.3

Conclusions:
The test material induced toxicity-related increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells with and without rat liver S9 microsomal activation. Only highly toxic treatments (survival rate 13% to 34%) in the 3 to 10 nl/ml concentration range induced approximately 2-to 4-fold incresses in the mutant frequency in the absence of S9 activation. With activation, similar increases were observed for moderately to highly toxic treatments (survival rate 20% to 60%) in the 25 to 75 nl/ml concentration range. The test material was therefore considered to be weakly active in the Mouse Lymphoma Forward Mutation Assay. The test material, C-150 (SN-2177), induced toxicity-related increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells with and without rat liver S9 microsomal activation.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Physical state: liquid
- Analytical purity: >95%
- Lot/batch No.: 030061P040
- Storage condition of test material: Room temperature, protected from light
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg/plate and in a second experiment 0, 1000, 2000, 3000, 4000 and 5000 µg/plate with strain TA 1535 only
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The test substance is completely soluble in DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S-9 mix for stains TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
without S-9 mix for strains TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
without S-9 mix for strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S-9 mix for strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S-9 mix for E.coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: the standard plate test was used


DURATION
- Exposure duration: 48-72 hours at 37°C

SELECTION AGENT (mutation assays): his+ and trp+ revertants

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity is detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp-
background growth), reduction in the titer is recorded for all test groups both with and without S-9 mix in all experiments.


Evaluation criteria:
Generally, the experiment is considered valid if the following criteria are met :
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data or above
- The titer of viable bacteria was >= 10 to the power of 8/ml.

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one
tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 500 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 500 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 500 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 500 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON GENOTOXICITY:
A non-dose dependent increase in the number of mutant colonies was observed for S. typhimurium strain TA1535 in both experiments only in the presence of S9 mix. The number of colonies increased from about 500 µg - 1 000 µg/plate (factor 1.7 - 1.8) onward with a maximum response at 2500 µg - 3000 µg/plate (factor 2.5 - 2.6), and a decreasing response at 4000 - 5000µg/plate (factor 2.0 - 2.1)

COMPARISON WITH HISTORICAL CONTROL DATA:
The controls were within the range of the included historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions at doses >= 500 µg/plate .

Table 1: Max. revertants per plate in the individual bacterial strains tested

Strain

Tested compound

Maximum revertants/plate [corresponding dose unit in µg/plate]

 

 

without S9-mix

with S9-mix

S. typhimurium TA1535

DMSO

16 ± 3

18 ± 1

Test substance

21 ± 4 [20]

46 ± 9 [2500]

Positive Control

802 ± 40 [5; MNNG]

114 ± 13 [2.5; 2-AA]

S. typhimurium TA100

DMSO

103 ± 5 

105 ± 5

Test substance

117 ± 7 [100]

102 ± 12 [20]

Positive Control

940 ± 67 [5; MNNG]

788 ± 85 [2.5; 2-AA]

S. typhimurium TA1537

DMSO

10 ± 3 

7 ± 2

Test substance

8 ± 1 [100]

8 ± 2 [20]

Positive Control

379 ± 32 [10; AAC]

123 ± 12 [2.5; 2-AA]

S. typhimurium TA98

DMSO

 28 ± 4

30 ± 4

Test substance

26 ± 4 [20]

30 ± 5 [20]

Positive Control

912 ± 39 [10; NOPD]

897 ± 76 [2.5; 2-AA]

E. coli WP2 uvrA

DMSO

29 ± 2

33 ± 3

Test substance

31 ± 4 [500]

32 ± 7 [500]

Positive Control

783 ± 80 [5; 4-NQO]

222 ± 16 [60; 2-AA]

S. typhimurium TA1535

DMSO

17 ± 2

17 ± 2

Test substance

18 ± 2 [1000]

45 ± 5 [3000]

Positive Control

940 ± 40 [5; MNNG]

132 ± 19 [2.5; 2-AA]

2-AA = 2-aminoanthracene  

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine  

NOPD = 4-nitro-o-phenylenediamine      

AAC = 9-aminoacridine      

4-NQO = 4-nitroquinoline-N-oxide      

Table 2: Detailed data on tester strain TA 1535

Without metabolic activation With metabolic activation
Revertants/plate Mean Factor Revertants/plate Mean Factor
DMSO 19 16 1 18 18 1
14 18
16 19
DMSO* 17 17 1 16 17 1
15 19
19 16
20µg 19 21 1,3 19 17 0,9
26 15
18 16
100µg 24 19 1,1 21 19 1,1
18 21
14 16
500µg 15 15 0,9 30 32 1,7
16 34
13 32
1000µg* 17 18 1 27 30 1,8
20 32
16 32
2000µg* 12 16 0,9 39 38 2,2
18 35
17 39
2500µg 15 16 1 49 46 2,5
18 54
15 36
3000µg* 14 15 0,9 46 45 2,6
16 40
14 49
4000µg* 16 15 0,9 37 34 2
13 34
15 31
5000µg 15 13 0,8 55 38 2,1
13 28
10 31
5000µg* 14 14 0,8 39 36 2,1
12 36
16 32
Positive control 839 802 49,1 104 114 6,2
758 109
811 129
Positive control* 902 940 55,3 140 132 7,8
982 111
937 146
*2nd experiment
Conclusions:
According to the results of the present study, the test substance is weakly mutagenic ín the Salmonella typhimurium strain TA 1535 with metabolic activation in this reverse mutation assay under the experimental conditìons chosen here .
Executive summary:

The substance was tested for its mutagenic potential based on the ability to induce point mufations ín selected loci of severa bacterial strains, i .e .Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, . TA 1537, TA 98 and E . coli WP2 uvrA

Dose range: 20 µg - 5 000 µg/plate

Test conditions: Standard plate test (SPT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix).

Solubility: No precipitation of the test substance was found

Toxicity: A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions at doses 500 µg/plate.

Mutagenicity:

TA 1535: Slight increase in the number of revertant colonies from about 500 µg - 1000 µg/plate (factor 1.7 - 1.8) onward with a maximum response at 2500 µg - 3000 µg/plate (factor 2.5 - 2.6) after the addition of a metabolizing system.

TA 100, TA 1537, TA 98, and E. coli WP2 uvrA: No increase in the number of his+ or trp+ revertants.

According to the results of the present study, the test substance is weakly mutagenic ín the Salmonella typhimurium reverse mutation assay under the experimental conditìons chosen here .

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Physical state: liquid
- Analytical purity: >90%
- Lot/batch No.: 88/1003
- Storage condition of test material: at 4-6°C
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The test substance is completely soluble in DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S-9 mix for strains: TA 100, TA 98, TA 1537 and TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitroso-guanidine (MNNG)
Remarks:
without S-9 mix for strains: TA 100 and TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
without S-9 mix for strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine chloride monohydrate
Remarks:
without S-9 mix for strain TA 1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: The standard plate test and the preincubation test were used

DURATION
- Preincubation period: 20 minutes
- Exposure duration: incubation for 48 hours at 37°C

SELECTION AGENT (mutation assays): his+ revertants

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
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:
see Additional information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
A slight decrease in the number of his+ revertants was observed in the standard plate test only with TA 98 at doses > 100 µg/plate.
In the preincubation assey a weakly bacteriotoxic effect was found depending on the strain and test conditions at doses > 2500 µg/plate
(TA 100, TA 1535) or at doses > 500 µg/plate (TA 98) .

Table 1: Maximum revertants/plate and corresponding test concentrations:

    

Strain

Tested compound

Maximum revertants/plate [corresponding dose unit in µg/plate]

1st experiment (Standard Plate Test)

 

without S9-mix

with S9-mix

S. typhimurium TA1535

DMSO

16 ± 5

25 ± 3

Test substance

28 ± 2 [2500]

30 ± 2 [2500]

Positive Control

1242 ± 597

315 ± 15

S. typhimurium TA100

DMSO

85 ± 8

121 ± 5

Test substance

111 ± 6 [500]

132 ± 14 [500]

Positive Control

1145 ± 83

1514 ± 72

S. typhimurium TA1537

DMSO

± 4

10 ± 3

Test substance

13 ± 6 [20]

13 ± 3 [500]

Positive Control

333 ± 30

180 ± 63 

S. typhimurium TA98

DMSO

21 ± 2

34 ± 3

Test substance

23 ± 1 [20]

35 ± 5 [20]

Positive Control

1310 ± 50

776 ± 13

2nd experiment (Preincubation Test)

 

without S9-mix

with S9-mix

S. typhimurium TA1535

DMSO

22 ± 4

18 ± 8

Test substance

26 ± 6 [500]

23 ± 8 [500]

Positive Control

1183 ± 50

237 ± 8

S. typhimurium TA100

DMSO

97 ± 9

101 ± 11

Test substance

109 ± 5 [2500]

120 ± 13 [500]

Positive Control

930 ± 61

1140 ±96 

S. typhimurium TA1537

DMSO

± 2

11 ± 1

Test substance

10 ± 1 [100]

13 ± 6 [100]

Positive Control

527 ± 47 

92 ± 6

S. typhimurium TA98

DMSO

20 ± 4

36 ± 5

Test substance

25 ± 3 [100]

38 ± 3 [500]

Positive Control

1210 ± 125 

847 ± 32

Conclusions:
An increase in the number og his+ revertants was not observed both in the standard plate test and in the pre-incubation test either without S9 mix or after the addition of a metabolizing system.
Executive summary:

The test substance was tested for mutagenicity in the Ames test.

Strains : TA 1535, TA 100, TA 1537, TA 98

Dose range : 20 µg - 5000 µg/plate

Test conditions : Standard plate test and preincubation test both with and without metabolic activation (rat liver S-9 mix) .

Solubility : Complete solubility of the test substance in DMSO .

Toxicity: A weakly bacteriotoxic effect was observed in the standard plate test only with TA 98 at doses100 µg/plate and in the

preincubation test at doses2500 µg/plate ( TA 1535, TA 100) or > 500 µg/plate ( TA 98) .

Mutagenicity: An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation

test either without S-9 mix or after the addition of a metabolizing system .

Assessment : According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here .

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
An Ames test was conducted according to the procedure described by Ames et al. 1975 (Mutation Res. 31: 347-364).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): C-150 SN-2177
- Physical state: colourless liquid
- Analytical purity: no data given
Target gene:
Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from the liver of male Sprague-Dawley rats, treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice and mixed with a series of cofactors.
Test concentrations with justification for top dose:
0.005, 0.01, 0.1, 1.0, 5.0, 10.0, 25.0, 50.0 µl per plate
Vehicle / solvent:
acetone
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: see "Details on test system and conditions"
Details on test system and experimental conditions:
Media:
The bacterial strains were cultured in Oxoid Media #2-(nutrient Broth). The selective medium was Vogel Bonner Medium E with 2% glucose. The overlay agar will consist of 0.6 % purified agar with 0.5 mM histidine, 0.05 mM biotin and 0.1 M NaCl.

Nonactivation Assay (without S9-mix):
To a sterile 13 x 100 mm test tube placed in a 43 °C water bath the following is added in order:
(a) 2.00 ml of 0.6 % agar containing 0.05 mM histidine and 0.05 mM biotin.
(b) 0.05 ml of a solution of the test chemical to give the appropriate dose.
(c) 0.1 ml - 0.2 ml of indicator organism/s.
(d) 0.50 ml of 0.01 M phosphate buffer, pH 7.4.
This mixture is swirled gently and then poured into minimal agar plates. After the top agar has set, the plates are incubated at 37 °C for approximately 2 days. The number of his+, revertant colonies growing on the plates is counted and recorded.

Activation Assay (with S9-mix):
The activation assay is run concurrently with the nonactivation assay. The only difference is the addition of 0.5 ml of S9 mix to the tubes in place of 0.5 ml of phosphate buffer which is added in nonactivation assays. All other details are similar to the procedure for nonactivation assays.

Control Compounds:
A negative control consisting of the solvent used for the test material is performed in all cases. For negative controls, step 'b' of Nonactivation Assays is replaced by 0.05 ml of the solvent. The negative controls are employed for each indicator strain and is performed in the absence and presence of S9 mix. The solvent used to prepare the stock solution of the test material is given in the Results section of this report. All dilutions of the test material made using this solvent. Specific positive control compounds known to revert each strain are also used in the assays:
Without S9-mix: Sodium azide (solvent water) 10 µg per plate for Salmonella strains TA1535 and TA100; 2-Nitrofluorene (NF) (solvent DMSO) 10 µg per plate for Salmonella strains TA1538 and TA98; 9-aminoacridine (9AA) (solvent ethanol) 50 µg per plate for Salmonella strains TA1537.
With S9-mix: 2-Anthramine (ANTH) (solvent DMSO) 2.5 µg per plate for all strains.
Evaluation criteria:
Evaluation criteria for mutagenicity:
- Strains TA-1535, TA-1537 and TA-1538:
If the solvent control value is within the normal range, a test material that produces a positive dose response over three concentrations with the highest increase equal to three times the solvent control value will be considered to be mutagenic.
- Strains TA-98 and TA-100:
If the solvent control value is within the normal range, a test material that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control va1ue for TA-98 and TA-100 will be considered to be mutagenic.
- Pattern:
Because TA-1535 and TA-100 are both derived from the same parental strain (G-46) and because TA-1538 and TA-98 are both derived from the same parental strain (03052), to some extent there is a built-in redundancy in the microbial assay. In general the two strains, of a set respond to the same mutagen and such a pattern is sought. Generally, if a strain responds to a mutagen in nonactivation tests, it will do so in activation tests.
- Reproducibility:
If a test material produces a response in a single test that cannot be reproduced in additional runs, the initial positive test data lose significance.

Evaluation Criteria for Toxicity:
- Complete toxicity:
When there are no revertants observed on the plate(s) treated with the test compound, the test compound will be defined as toxic to all or any of the indicator strains at that particular dose(s).
- Slight toxicity:
When there are fifty per cent or less number of revertants on the plate(s) treated with the test compound as compared to the solvent control plate(s), the test compound will be defined as slightly toxic to all or any of the indicator strains at that particular dose(s).
Species / strain:
other: S. typhimurium TA 1535, TA 1537, Ta1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: see "Additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY:
The test material was toxic to the strains TA-1537 and TA-1538 at 10 µl per plate and to TA-100 at 25 µl and 50 µl per plate. The test material was also slightly toxic at 10 µl per plate for TA-1535 and at 25 µl and 50 µl per plate for the strain TA-98.

MUTAGENICITY:
The results of the tests conducted on the test material in the absence of a metabolic activation system were negative. The results of the tests conducted on the test material in the presence of a rat liver activation system were also negative.

Table 1: Maximum revertants/plate and corresponding test concentrations:

Strain

Tested compound

Maximum revertants/plate [corresponding concentrations in µl/plate]

 

 

without S9-mix

with S9-mix

S. typhimurium TA1535

DMSO

7

14

Test substance

13 [0.1]

14 [1.0]

Positive Control

855

162

S. typhimurium TA1537

DMSO

4

8

Test substance

6 [0.005]

9 [1.0]

Positive Control

263

168

S. typhimurium TA1538

DMSO

11

47

Test substance

13 [0.01]

57 [0.1]

Positive Control

1482

1227

S. typhimurium TA98

DMSO

31

34

Test substance

32 [5.0]

56 [0.01]

Positive Control

1099

1171

S. typhimurium TA100

DMSO

222

238

Test substance

248 [0.01]

254 [0.1]

Positive Control

1531

254

Conclusions:
Under the experimental conditions chosen, the test material was not mutagenic in the Ames test (S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100) with or without the addition of metabolic activation S9-mix.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

in vivo Micronucleus Test: negative - not clastogenic or aneugenic

- OECD 474; GLP; NMRI mouse, i.p.; 87.5, 175, and 350 mg/kg bw/day: not clastogenic or aneugenic (2004)

- OECD 474; GLP; CD1 mouse, oral up to 2000 mg/kg bw/day: not clastogenic or aneugenic (2007)


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Physical state: liquid
- Analytical purity: >95%
- Lot/batch No.: 030061P040
- Stability under test conditions: the stability of the test substance throughout the study period and in the vehicle was verified analytically.
- Storage condition of test material: room temperature, protected from light
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 31 g (mean)
- Assigned to test groups randomly: yes, under following basis: randomization plan prepared with an appropriate computer program.
- Housing: Makrolon cages, type MI, housed individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, olive oil was selected as the vehicle, which had
been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 0.875 g/100 ml; 1.75 g/100 ml and 3.5 g/100 ml
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in olive oil:
- The low dose group was given 87.5 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of
0.875 g/100 ml.
- The intermediate dose group was given 175 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of
1.75 g/100 ml.
- The top dose groups were given 350 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 3.5 g/100 ml.
Duration of treatment / exposure:
one single administration
Frequency of treatment:
one single administration
Post exposure period:
24-48 hours
Dose / conc.:
87.5 mg/kg bw/day (nominal)
Dose / conc.:
175 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPP) and vincristine (VCR) both dissolved in purified water were administered to male animals once intraperitoneally each in a
volume of 10 ml/kg body weight.
- Justification for choice of positive control(s): The stability of CPP and VCR is well-defined under the selected conditions, since both positive control
articles are well-established reference clastogens and aneugens respectively.
- Route of administration: intraperitoneal
- Doses / concentrations: CPP: 20 mg/kg bw for clastogenic effects; VCR: 0.15 mg/kg bw for aneugenic effects
Tissues and cell types examined:
In general, 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored .
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of
350 mg/kg body weight and in the vehicle controls. In the test groups of 175 mg/kg and 87.5 mg/kg body weight and in the positive control groups,
the 24-hour sacrifice interval was investigated only.

DETAILS OF SLIDE PREPARATION:
The two femora were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the
diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur). The suspension was mixed
thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified
water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes.
After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an
index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression
may be identified and excluded from the evaluation.
- Number of small micronuclei (d=D/4) (d = diameter of micronucleus, D= cell diameter)
The size of micronuclei may indicate the possible mode of action of the test substance, i .e . a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.
Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative
values can occur.
Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i .e. >=2000 polychromatic erythrocytes and a clear differentiation between polychromatic erythrocytes (PECs) and normochromatic erythrocytes (NECs).
-The ratio of PECs/NECs in the untreated animals (negative control) has to be within the normal range of the animal strain.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for
PECs and NECs.
- The two positive control substances have to induce a significant increase in the number of PECs containing small and large micronuclei within the
range of the historical control data or above.

A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.

A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN.
The asymptotic U test according to Mann-Whitney (modified rank test according to Wilcocon) was carried out to clarify the question whether there were significant differerences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes.
The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determinatian for the U test .
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
the test substance led to clinical signs
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

As a negative control, male mice were administered merely the vehicle, olive oil,by the same route, which

gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.
Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity.

The administration of the test substance led to clinical signs, namely piloerection and squatting posture.

According to the results of the present study, the single intraperitoneal administration of Tripropylenglykoldiacrylat did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data.

A dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected from about of 87.5 mg/kg body weight onward.

Conclusions:
Thus, under the experimental conditions chosen in the study, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): TPGDA
- Analytical purity: approx. 87 %
- Lot/batch No.: P8960464SAP
- Expiration date of the lot/batch: 19 March 2008
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, USA
- Age at study initiation: Young adults, approx. 8 weeks old
- Weight at study initiation: males: 33.7 to 38.2 g; females: 23.1 to 28.1 g
- Assigned to test groups randomly: yes by using a computer program
- Housing: separated by gender, up to 5 animals per cage
- Diet: PMI Certified Rodent Diet (R) #5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17.7 - 26.1 °C
- Humidity: 30-70 %
- Air changes per hr: >/= 10
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil (CAS No. 8001-30-7); Supplier: Welch, Holme & Clarke; Lot No. 12-455; Storage at > 0 °C to 10 °C
Details on exposure:
Dose Preparation
Prior to dosing, the top stock of the test article, TPGDA, was prepared by adding the appropriate volume of the vehicle, corn oil, to a pre-weighed quantity of the test article and mixed, forming a solution. Lower concentrations were obtained by dilution with the vehicle. The formulations were held at room temperature prior to dosing.

Dose Analyses
The Sponsor was responsible for the determination and documentation of the identity, strength, purity, stability and uniformity of the test article and the determination of stability, homogeneity and concentration of the dosing preparations.


Duration of treatment / exposure:
one single administration
Frequency of treatment:
one single administration
Post exposure period:
24 h for all dose groups; additionally 48 h for an additional vehicle control and an additional 2000 mg/kg bw group
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Range-finder and main study
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Range-finder and main study
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Range-finder and main study
No. of animals per sex per dose:
Range-finding study: 3 males and 3 females per dose
Main study: 5 males per dose and harvest time point
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CAS No. 6055-19-2); Supplier: Sigma Aldrich; Lot No. 076K1050; Storage in a refrigerator set to maintain >0 to 10 °C;
the solvent was water
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Extraction of Bone Marrow
The hind limb bones (tibias) were removed for marrow extraction from five surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).

Preparation of Slides
Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol, stained in May-Grünwald solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides were coded prior to analysis.

Slide Analysis
Slides prepared from the bone marrow collected from five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least 500 erythrocytes per animal.
Evaluation criteria:
Acceptable Controls
The vehicle control group mean must lie within the historical control range and will usually be less than 0.4% micronucleated PCEs. There must be a statistically significant elevation of the mean of the positive control group relative to the vehicle control group, and the positive control response must be consistent with historical positive control data.
Acceptable High Dose
Generally the high dose should reach the limit dose or produce some indication of toxicity, e.g., toxic signs and/or mortality in the test article dosed animals and/or a reduction in the PCE:NCE ratio. If there are solubility constraints, the highest dose tested will be the solubility limit or higher doses if a well-dispersed suspension is obtained that does not settle out rapidly.
Assay Evaluation Criteria
The criteria for a positive response is the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose related response. A test article that does not induce both of these responses is considered negative. Statistical significance is not the only determinant of a positive response; the Study Director also considers the biological relevance of the results in the final evaluation.

Statistics:
The following statistical methods were used to analyze the micronucleus data.
• Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances.
• If the analysis of variance was statistically significant (p ≤ 0.05), Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time.
The 500, 1000, and 2000 mg/kg dose groups, as well as the positive control group, were compared with the vehicle control group at the 5% probability level.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose Range-finding Study:
Survival and Clinical Observations:
All animals appeared normal immediately after dosing and remained healthy until the end of the observation period.
Conclusion:
The high dose of the commercial product reached the maximum allowable dose of 2000 mg/kg bw, based on regulatory guidelines.


Micronucleus Assay:
- Survival and Clinical Observations:
All animals in all the dose groups appeared normal immediately after dosing and remained healthy until the appropriate harvest timepoint. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest timepoint.
- Results and Interpretation:
A commercial product of TPGDA did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (500, 1000, or 2000 mg/kg). TPGDA was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose of the test article analyzed.
The vehicle control group had approximately ≤0.09% micronucleated PCEs and the group mean was within the historical control range. The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard error of 1.93 ± 0.21%.

Table 1: Individual animal data after 24 and 48 h treatment

Treatment

Dose

Animal No.

# MN PCE/2000 PCE

Ratio PCE:NCE

vehicle control 24 h

corn oil 10 ml/kg bw

1

1

0.29

 

corn oil 10 ml/kg bw

2

2

0.26

 

corn oil 10 ml/kg bw

3

2

0.66

 

corn oil 10 ml/kg bw

4

1

0.25

 

corn oil 10 ml/kg bw

5

3

0.46

vehicle control 48 h

corn oil 10 ml/kg bw

1

3

0.56

 

corn oil 10 ml/kg bw

2

1

0.57

 

corn oil 10 ml/kg bw

3

3

0.31

 

corn oil 10 ml/kg bw

4

1

0.26

 

corn oil 10 ml/kg bw

5

1

0.50

positive control

cyclophosphamide 80 mg/kg bw

1

50

0.10

 

cyclophosphamide 80 mg/kg bw

2

26

0.36

 

cyclophosphamide 80 mg/kg bw

3

45

0.52

 

cyclophosphamide 80 mg/kg bw

4

38

0.74

 

cyclophosphamide 80 mg/kg bw

5

34

0.57

Test material 24 h

500 mg/kg bw

1

2

0.81

 

500 mg/kg bw

2

0

0.44

 

500 mg/kg bw

3

0

0.56

 

500 mg/kg bw

4

0

0.38

 

500 mg/kg bw

5

3

0.20

Test material 24 h

1000 mg/kg bw

1

1

0.30

 

1000 mg/kg bw

2

0

0.49

 

1000 mg/kg bw

3

0

0.76

 

1000 mg/kg bw

4

1

0.42

 

1000 mg/kg bw

5

1

0.64

Test material 24 h

2000 mg/kg bw

1

0

0.32

 

2000 mg/kg bw

2

1

0.48

 

2000 mg/kg bw

3

4

0.29

 

2000 mg/kg bw

4

6

0.45

 

2000 mg/kg bw

5

1

0.53

Test material 48 h

2000 mg/kg bw

1

0

0.53

 

2000 mg/kg bw

2

1

0.36

 

2000 mg/kg bw

3

1

0.45

 

2000 mg/kg bw

4

2

0.31

 

2000 mg/kg bw

5

1

0.26

PCE = Polychromatic erythrocyte

MN PCE = Micronucleated PCE

NCE = Normochromatic erythrocyte

Conclusions:
The test material was evaluated as negative in the mouse bone marrow micronucleus assay under the experimental conditions of this assay chosen.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Valid experimental data were available to assess the genetic toxicity in vitro and in vivo - based on the outcome of those tests TPGDA is giving no concern in respect to a mutagenic or clastogenic potential and no additional animal tests are deemed necessary.

Reliable in vitro studies gave no indication of gene mutations in bacteria (Ames test) or mammalian cells (HPRT assay). Only at high cytotoxic doses the test substance was tested positive in a mouse lymphoma assay using L5178Y cells, this weak potential was considered to originate from cytotoxicity inducing clastogencity. No clastogenic effect and no aneugenic activity was detected in valid in vivo micronucleus tests after i.p. and oral administration. In vitro, no gene mutations were detected in bacteria and mammalian cells, therefore no additional animal tests (i.e. mammalian alkaline comet assay of trangenic rodent somatic and germ cell gene mutation assay) are needed to assess the endpoint genetic toxicity

IN VITRO

Gene mutation in bacteria: not mutagenic

The test substance was tested for mutagenicity in an Ames test with and without metabolic activation, at 0, 20, 100, 500, 2500 and 5000 µg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98, TA 100, and in E. coli WP2 uvrA and in a second experiment at 0, 1000, 2000, 3000, 4000 and 5000 µg/plate in TA 1535 only; (acc. OECD 471, 2003). An increase in the number of mutant colonies that lacked dose dependency was observed only in the strain S. typhimurium TA1535 after addition of S9 mix from about 500 µg - 1000 µg/plate (factor 1.7 - 1.8) onward with a maximum response at 2500 µg - 3000 µg/plate (factor 2.5 - 2.6) and a reduced mutation rate (factor 2 - 2.1) at higher concentrations. Cytotoxicity was observed at 500 µg/plate and higher, depending on test strain.

 

In another standard plate Ames test with and without metabolic activation (tested at 0, 20, 100, 500, 2500 and 5000 µg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98 and TA 100, the test substance was not mutagenic. Metabolic activation was from S9 fraction from the liver of rats, treated with Aroclor 1254 (acc. OECD 471, 1989). A slight decrease in the number of his+ revertants was observed in the standard plate test only with TA 98 at doses > 100 µg/plate. In the preincubation assay a weakly bacteriotoxic effect was found depending on the strain and test conditions at doses > 2500 µg/plate (TA 100, TA 1535) or at doses > 500 µg/plate (TA 98).

 

A third Ames test also provided negative results for the test substance with or without metabolic activation, tested in S. typhimurium strains TA 1535, TA 1537, 1538, TA 98 and TA 100, tested at concentrations of 0.005, 0.01, 0.1, 1.0, 5.0, 10.0, 25.0, 50.0 µl per plate (Litton Bionetics Inc. 1980, Val. 2). The test material was toxic to the strains TA-1537 and TA-1538 at 10 µl per plate and to TA-100 at 25 µl and 50 µl per plate. The test material was also slightly toxic at 10 µl per plate for TA-1535 and at 25 µl and 50 µl per plate for the strain TA-98.

 

In conclusion, the test substance was weakly positive in one of three Ames tests in a single tester strain (TA1353) with metabolic activation only. The increase in revertant colonies was at most a factor of 2.6 above concurrent negative controls and lacked a dose response relationship. In combination with two further Ames assays tested at equally high or even higher concentrations, which showed no positive response in all tester strains with or without metabolic activation, the test substance is not considered to cause gene mutations in bacteria.

 

 

Gene mutation in mammalian cells: WoE not mutagenic

To exclude that the test substance has the potential to cause gene mutations in mammalian cells, an HPRT assay according to OECD and GLP guidelines was performed (2015). Chinese hamster ovary cells (CHO) were treated with up to 30µg/mL without S9 mix and up to 140µg/mL after the addition of a metabolizing system. Due to cytotoxicity, only concentrations up to 8.74µg/mL without S9 and 60µg/mL with S9 could be evaluated. No biologically relevant increase in the mutant frequency was observed in two repeated experiments. The test substance is thus considered non-mutagenic in mammalian cells.

Only at high cytotoxic doses the test substance was tested positive for genotoxicity in a mouse lymphoma assay using L5178Y cells at levels up to 12.5 nl/ml without metabolic activation and up to 75 nl/ml with metabolic activation (1980, Val. 2). The test material induced toxicity-related increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells with and without rat liver S9 microsomal activation. Only highly toxic treatments (survival rate 13% to 34%) in the 3 to 10 nl/ml concentration range induced approximately 2-to 4-fold increases in the mutant frequency in the absence of S9 activation. With activation, similar increases were observed for moderately to highly toxic treatments (survival rate 20% to 60%) in the 25 to 75 nl/ml concentration range. It was concluded that the test material induced toxicity-related increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells with and without rat liver S9 microsomal activation.

However, several arguments can be presented to refute the apparent positive outcome of the mouse lymphoma assay, taking into account results of other multifunctional acrylates. Firstly, no colony sizing was performed to differentiate between gene mutations and clastogenic activity.Whenever colony sizing was performed in a mouse lymphoma assay with other multifunctional acrylates, the prevalence of small colonies indicates clastogenicity in vitro. Secondly, results obtained from several multifunctional acrylates follow a common pattern of positive results in in vitro clastogenicity assays only. In vivo clastogenic studies produced always negative results. Positive results in in vitro clastogenicity assays are presumably due to high cytotoxicity of test materials, which is also true for the test substance in question, inducing an increase in mutant frequency only at cytotoxic concentrations in this mouse lymphoma assay (Mutagenicity assessment of acrylate and methacrylate compounds and implications for regulatory toxicology requirements, Johannsen et al, Regulatory Toxicology and Pharmacology 50 (2008) 322–335). Additionally, dose-response curves for cell survival are very steep, which means that even a minor increase in the concentration will drastically reduce the number of surviving cells. Taken all information together, the weak mutagenic potential observed in the mouse lymphoma assay was considered to originate from cytotoxicity rather than from a true mutagenic effect of the test substance.

In conclusion TPGDA did not induce gene mutations in bacteria and mammalian cells. Only in high cytotoxic concentrations an increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells was described,

no colony sizing was performed to differentiate between gene mutations and clastogenic activity. But whenever colony sizing was performed in a mouse lymphoma assay with other multifunctional acrylates, the prevalence of small colonies indicates clastogenicity.

IN VIVO

The test substance showed no mutagenic activity in in vivo assays with rodents. In a mouse micronucleus assay on polychromatic erythrocytes, the test substance yielded a negative result after single oral administration of 87.5, 175, 350 mg/kg bw. Sampling times were 24 and 48 h (2004, Val. 1). Male mice were administered merely the vehicle, olive oil, by the same route serving as negative control. Frequencies of micronucleated polychromatic erythrocytes were within the historical control range. Both positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Animals which were administered the vehicle, or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. The administration of the test substance led to clinical signs, namely piloerection and squatting posture. According to the results of the present study, the single intraperitoneal administration of the test substance did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. A dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected from about of 87.5 mg/kg body weight onward. Thus, under the experimental conditions chosen in the study, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

In a supporting in vivo micronucleus study, the test substance was also found to be negative after oral administration. In this test, doses of 2000, 1000 and 500 mg/kg bw were administered. Sampling time was 24 and 48 h after administration. Five males per dose and harvest time point were used (2007, Val. 1). All animals in all dose groups and controls appeared normal immediately after dosing and remained healthy until the appropriate harvest timepoint. The test substance did not induce statistically significant increases in micronucleated PCEs at any dose examined (500, 1000, or 2000 mg/kg). The test substance was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose analyzed. The vehicle control group had approximately0.09% micronucleated PCEs and the group mean was within the historical control range. The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control. In conclusion, the test substance was considered not to be clastogenic.

In another supporting study, the test substance was applied dermally to Tg.AC mice (3 times a week for 20 weeks). Peripheral blood leukocytes were evaluated for DNA damage (single-strand breaks, alkali labile sites, DNA crosslinking) at weeks 4, 8, 12, 16, and 20 by using the alkaline (pH > 13) single cell gel (SCG) assay. Peripheral blood polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were evaluated for the presence of micronuclei at week 20 (Tice 1997, Val. 2). The extent of DNA migration in leukocytes of mice treated by dermal application with the test substance at 1, 5, or 10 μmol per mouse was not significantly different, either by trend test analysis or by a pairwise comparison of each treatment dose against the concurrent vehicle control, at any sample time. TPA (0.002 μmol per mouse), the positive control for the tumorigenicity studies, also failed to significantly alter the extent of DNA migration or its intercellular dispersion in leukocytes of mice treated by dermal application. After 20 weeks of treatment, the frequency of micronucleated PCE and NCE in blood were not increased in the mice treated with the test substance or TPA. The percentage of PCE was increased in mice treated with the test substance which was highly significant. By a pairwise comparison, the lowest effective dose of the test substance inducing a significant increase in percentage of PCE was 10 μmol per mouse. TPA, at 0.002 μmol per mouse also induced a marginally nonsignificant increase in the percentage of PCE. This observed increase in the rate of erythropoiesis may reflect bone marrow/blood toxicity, a homeostatic mechanism in response to the treatment-induced tumor burden, and/or a hematopoietic response to epidermal keratinocyte cytokines induced by tissue injury. The authors concluded that in this study, the dermal application of the test substance to female Tg.AC mice over a 20-week period, failed to induce a significant increase in DNA damage in circulating leukocytes at multiple sample times or chromosomal damage in proliferating bone marrow cells. 

In conclusion, the test substance did not induce chromosome damage or DNA damaging in in vivo genotoxicity studies.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) 2018/1480, classification as a mutagen is not warranted.