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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames assay (OECD 471) revealed to be negative, whilst two other in vitro genotoxicology assays, a mouse lymphoma assay (OECD 490) and an micronucleus (OECD 487) were found to be positive

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2015-08-05 to 2015-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study, OECD 471 compliant
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Remarks:
2013-05-06
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Dose range finding 1: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate without and with metabolic activation (TA 100 and WP2uvrA; direct plate assay)
Experiment 1: 5.4, 17, 52, 164, 512 and 1600 μg/plate without and with metabolic activation (TA1535, TA1537 and TA98; direct plate assay)
Dose range finding 2: 1.7, 5.4, 17, 52, 164, 512 and 1600 µg/plate without and with metabolic activation (TA 100 and WP2uvrA; pre-incubation assay)
Experiment 2: 5.4, 17, 52, 164, and 512 μg/plate without and with metabolic activation (TA1535, TA1537 and TA98; pre-incubation assay)
Vehicle / solvent:
Milli-Q water (Millipore Corp., Bedford, MA., USA)
Untreated negative controls:
yes
Remarks:
Milli-Q water (Millipore Corp., Bedford, MA., USA)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191
Remarks:
Solvents for reference items Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands) DMSO = dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany)
Details on test system and experimental conditions:
Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria

Source: Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames) (TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006) and (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) (WP2uvrA, 2008)

Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Cell culture
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Merck) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Merck) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Acros Organics).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Merck) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 32.4 – 38.9°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Any variation were evaluated and maintained in the raw data.

All the test were evaluated in triplicate.
Evaluation criteria:
Acceptability of the assay

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Data evaluation and statistical procedures

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
No formal hypothesis testing was done.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Water solubility: the substance was soluble at all concentration tested
- Precipitation: no precipitation observed


RANGE-FINDING/SCREENING STUDIES: the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. Toxicity was observed in both tester strains.

In the second mutation experiment, the test item was initially tested up to concentrations of 1600 µg/plate in the strains TA100 and WP2uvrA in the pre-incubation assay. Toxicity was observed in both tester strains.


COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Dose range finding test (direct incorporation test): Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with
one strain of Salmonella typhimurium and one Escherichia coli strain.

TA 100

WP2uvrA

Without S9 mix

Positive control

1061

±

42

1347

±

118

Solvent control

100

±

9

28

±

4

1.7

96

±

8

27

±

6

5.4

88

±

22

22

±

8

17

94

±

16

28

±

4

52

94

±

12

28

±

1

164

98

±

10

n

33

±

4

n

512

73

±

6

m

17

±

3

s

1600

0

±

0

a

0

±

0

a

5000

0

±

0

a NP

0

±

0

a NP

 

With S9 mix

Positive control

1456

±

105

402

±

18

Solvent control

106

±

24

32

±

7

1.7

89

±

24

25

±

2

5.4

105

±

25

27

±

7

17

86

±

4

25

±

8

52

90

±

7

30

±

1

164

87

±

8

n

26

±

5

n

512

84

±

16

m

22

±

5

s

1600

0

±

0

a

0

±

0

a

5000

0

±

0

a NP

0

±

0

a NP

NP         No precipitate

a             Bacterial background lawn absent

m           Bacterial background lawn moderately reduced

n            Normal bacterial background lawn

s             Bacterial background lawn slightly reduced

Experiment 1: Mutagenic response in the Salmonella typhimurium reverse mutation assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with

different strains of Salmonella typhimurium.

TA 1535

TA 1537

TA 98

Without S9 mix

Positive control

779

±

26

621

±

80

1513

±

41

Solvent control

8

±

3

4

±

1

26

±

7

5.4

11

±

4

5

±

0

22

±

3

17

12

±

3

7

±

4

16

±

2

52

9

±

6

8

±

3

26

±

14

164

14

±

5

n

9

±

2

n

29

±

3

n

512

8

±

3

s

10

±

5

s

19

±

6

s

1600

0

±

0

a NP

0

±

0

a NP

0

±

0

a NP

 

With S9 mix

Positive control

287

±

13

453

±

27

1203

±

34

Solvent control

10

±

2

6

±

1

34

±

8

5.4

8

±

3

6

±

2

27

±

2

17

13

±

6

7

±

4

30

±

4

52

10

±

2

8

±

3

30

±

4

164

11

±

6

n

6

±

1

n

37

±

5

n

512

10

±

0

s

7

±

4

s

40

±

9

s

1600

0

±

0

a NP

0

±

0

a NP

0

±

0

a NP

NP

No precipitate

a

Bacterial background lawn absent

n

Normal bacterial background lawn

s

Bacterial background lawn slightly reduced

Toxicity in the dose range finding test/first experiment ( Direct plate assay)

Strain

Without S9-mix

With S9-mix

             Dose         Bacterial                    Revertant

             (μg/plate)  background lawn       colonies

Dose         Bacterial                     Revertant

(µg/plate)  background lawn        colonies

Dose range finding test

TA100

512       moderate                  -1

1600     absent                  complete

5000     absent                  complete

512            moderate                       -1

1600          absent                     complete

5000          absent                      complete

WP2uvrA

512       slight                           -1

1600     absent                  complete

5000     absent                   complete

512             slight                               -1

1600          absent                      complete

5000          absent                      complete

First mutation experiment

TA1535

512       slight                         -1

1600     absent                  complete

512            slight                             -1

1600          absent                        complete

TA1537

512       slight                         -1

1600     absent                  complete

512            slight                             -1

1600          absent                        complete

TA98

512       slight                         -1

1600     absent                  complete

512            slight                             -1

1600          absent                        complete

1         No reduction in the number of revertant colonies less than the minimal value of the historical control data range.

Conclusions:
Interpretation of results (migrated information):
negative both in the absence and presence of S9-metabolic activation.

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Phosphonium, tetrakis (hydroxymethyl)-sulphate (2:1); polymer with urea is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Phosphonium, tetrakis (hydroxymethyl)-sulphate (2:1); polymer with urea was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254).

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

 

In the dose range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. Toxicity was observed in both tester strains. In the first mutation experiment, the test item was tested up to concentrations of 1600 µg/plate in the strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains.

 

In the second mutation experiment, the test item was initially tested up to concentrations of 1600 µg/plate in the strains TA100 and WP2uvrA in the pre-incubation assay. Toxicity was observed in both tester strains. In the second part of the experiment, the test item was tested up to concentrations of 512 µg/plate in the strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains.

 

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Phosphonium, tetrakis (hydroxymethyl)-sulphate (2:1); polymer with urea did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that Phosphonium, tetrakis (hydroxymethyl)-sulfate (2:1); polymer with urea is not mutagenic in the Salmonella typhimurium reverse assay and in Escherichia coli reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
yes
Remarks:
seven out of eight plates in dose 25µg/L and 40 µg/L
GLP compliance:
yes (incl. QA statement)
Remarks:
2015-11-02
Type of assay:
other: Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells
Target gene:
thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: L5178Y TK+/- cells, were obtained from ATCC (American Type Culture Collection, Manassas, USA), through Biovalley (Marne-La-Vallée, France).
-The cells were stored in a cryoprotective medium [10% horse serum and 10% dimethylsulfoxide (DMSO)] at -80°C. Each batch of frozen cells was purged of spontaneous TK-/- mutants and checked for the absence of mycoplasma. The cells were maintained in flasks as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 10% v/v, in a 37°C, 5% CO2 humidified incubator.

MEDIA USED
- The cells were stored in a cryoprotective medium [10% horse serum and 10% dimethylsulfoxide (DMSO)] at -80°C. Each batch of frozen cells was purged of spontaneous TK-/- mutants and checked for the absence of mycoplasma. The cells were maintained in flasks as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 10% v/v, in a 37°C, 5% CO2 humidified incubator.

- The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL).
This medium (RPMI 0) was supplemented by heat inactivated horse serum at 10%, v/v (RPMI 10) or 20% v/v (RPMI 20).
RPMI 10 was diluted with RPMI 0 (1:1, v/v) in order to obtain RPMI 5 which was used for the 3 hour treatment.


CULTURE CONDITIONS
- 37°C, 5% CO2 humidified incubator
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, consists of induced enzymatic systems contained in rat liver microsomal fraction (S9 fraction) Moltox INC, Boone, NC 28607, USA
Test concentrations with justification for top dose:
The selected dose levels were 1.88, 3.75, 7.5, 15, 25, 40, 60 and 100 µg/mL, both with and without S9 mix.
In the absence of S9 mix, a slight to severe cytotoxicity was induced at dose levels ≥ 40 µg/mL, as shown by a 59 to 99% decrease in the Adj. RTG. The recommended level of cytotoxicity was reached at the dose-level of 60 µg/mL (mean Adj. RTG of 14%).
In the presence of S9 mix, a moderate to severe cytotoxicity was induced at dose levels ≥ 60 µg/mL, as shown by a 62 to 96% decrease in the Adj. RTG. None of the selected dose levels induced the recommended level of cytotoxicity. However, considering the narrow dose levels spacing used, this selection was considered as suitable to allow a reliable interpretation.
Vehicle / solvent:
According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control cultures was water for injections.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding : 5 10e5

DURATION
- Exposure duration: 3 hour treatment period,in the absence as well as in the presence of S9-mix.
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): 7 days for the vialability plate and 11-12 days for the mutant plate

SELECTION AGENT (mutation assays): TFT (trifluorothymidine)


NUMBER OF REPLICATIONS:
Viability paltes: 2 cultures and 2 plates per culture / dose level
Mutants paltes: 2 cultures and 3 or 4 plates per culture / dose level

NUMBER OF CELLS EVALUATED:
Viability plate: an average of 1.6 cells/well in a 96-well plate
Mutant plate: 2000 cells/well in a 96-well plate

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity was evaluated by the calculation of the Relative Total Growth (RTG)

ACCEPTANCE CRITERIA
Each mutagenicity experiment (with or without S9 mix) is considered valid if the following criteria are met:
- At least four analyzable dose levels, i.e. having a mean Adj. RTG ≥ 10%, should be available,

- For the vehicle control:
- The mean Cloning Efficiency at the end of the expression time (CE2) should be between 0.65 and 1.2,
- The mean Mutation Frequency (MF) should falls within the normal range of 50 x 10-6 - 170 x 10e-6,
- Tthe mean Suspension Growth (SG) should be between 8 and 32.
- For the positive control:
- The upper limit of cytotoxicity observed in the positive control culture should have an Adj. RTG greater than 10%,
- There should be:
- Either an increase of the MF above the vehicle control (IMF) of at least 300 x 10-6, the increase in the small colony mutation frequency accounting for at least 40%,
- Or an increase in the small colony mutation frequency of at least 150 x 10-6 above that seen in the concurrent vehicle control.
Unless a positive result is obtained in at least one of the tested conditions, the study is considered valid if two experimental conditions were tested (short treatment with and without S9 mix)
Evaluation criteria:
In all cases, biological relevance is taken into consideration when evaluating the results.

Evaluation of a positive response:
Based on IWGT recommendations, a test item is considered clearly positive if, in any of the experimental conditions examined:
- At least at one dose level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10 e 6,
- A dose-response relationship is demonstrated by a statistically significant trend test.

Evaluation of a negative response:
A test item is considered clearly negative if, in all experimental conditions, no dose-response relationship is demonstrated or, if there is an increase in MF, it does not exceed the GEF.

Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose levels with Adj. RTG between 10 and 20%, are not considered as positive results.

A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if :
- There is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- There is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.
Statistics:
To assess the dose-response relationship, a linear regression was performed between dose levels and individual mutation frequencies obtained at dose levels showing a mean Adj. RTG ≥ 10% (see Appendix 4). This statistical analysis was performed using SAS Enterprise Guide software.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Preliminary toxicity test

Doses µg/mL(1)  Post-treat.
cell count*
Day 0
Adj. Conc.* Cell
count*
Day 1
Adj. Conc.* Cell
count*
Day 2
Cell
Count
factor
SG Adj.
RSG
%
Empty wells # CE2 RCE2      %      Adj.
RTG
%
Decrease in Adj. RTG
Without
 S9-mix
(3-hours)
0   2.68 2.00 7.15 2.00 6.70 1.0 12.0 100 22 27 0.85 100 100  
10   2.95 2.00 6.50 2.00 6.10 1.1 9.9 91 21 21 0.95 111 101 none
100   2.22 2.00 1.03 1.03 0.87 0.8 0.4 3 62 61 0.28 33 1 99
500   2.52 2.00 2.78 2.00 2.00 0.9 1.4 11 95 95 0.01 1 0 100
1000 P 2.34 2.00 2.95 2.00 1.90 0.9 1.4 10 95 96 0.00 0 0 100
2500 P 0.80 0.80 0.24 0.24 0.54 0.3 0.7 2 96 96 0.00 0 0 100
5000 P - - - - - - - - - - - - - -
With
 S9-mix
(3-hours)
0   2.00 2.00 8.05 2.00 6.85 1.0 13.8 100 21 25 0.89 100 100  
10   1.66 1.66 5.50 2.00 5.85 0.8 9.7 58 18 14 1.12 125 73 27
100   2.75 2.00 1.72 1.72 2.28 1.4 1.1 11 63 59 0.28 32 4 96
500   1.51 1.51 2.22 2.00 2.24 0.8 1.6 9 96 94 0.01 1 0 100
1000 P 1.51 1.51 3.08 2.00 2.00 0.8 2.0 11 96 96 0.00 0 0 100
2500 P 0.44 0.44 0.19 0.19 0.14 0.2 0.3 1 96 96 0.00 0 0 100
5000 P 0.00 - - - - - - - - - - - - -

*: x 105cells/mL

Adj. conc.: adjusted cell concentration

SG: suspension growth

Adj. RSG: adjusted relative suspension growth

CE2: cloning efficiency

RCE2: relative cloning efficiency

Adj. RTG: adjusted relative total growth

#: counted on a total number of 96 wells

0: vehicle control (Water for injections)

(1):expressed as active item

 -: without S9 mix: not evaluated due to the severe precipitate observed

 -: with S9 mix: not evaluated due to the severe toxicity observed

P: precipitate observed in the culture medium at the end of treatment

Main experiment without S9 mix, 3 hours treatment: cytotoxicity results

Doses µg/mL(1)   Post-treat.
cell count*
Day 0
Adj. Conc.* Cell
count*
Day 1
Adj. Conc.* Cell
count*
Day 2
Cell
count
factor
SG Adj.
RSG
%
Empty wells # CE2 RCE2% Adj.
RTG
%
Mean Adj. RTG % Decrease in Adj. RTG %
0 C1 3.10 2.00 5.85 2.00 6.80 1.0 9.9 100 18 19 1.03 100 100 100  
C2 1.97 1.97 6.55 2.00 5.60 1.0 9.3 100 26 10 1.05 100 100
1.88 C1 2.73 2.00 6.10 2.00 7.30 0.9 11.1 99 21 31 0.82 79 78 107 None
C2 3.08 2.00 6.00 2.00 4.60 1.6 6.9 116 10 17 1.23 117 136
3.75 C1 2.88 2.00 5.25 2.00 6.05 0.9 7.9 74 19 18 1.03 100 74 84 16
C2 2.55 2.00 4.50 2.00 5.30 1.3 6.0 83 17 12 1.18 113 94
7.5 C1 2.58 2.00 5.05 2.00 7.25 0.8 9.2 77 14 18 1.12 109 83 98 2
C2 2.68 2.00 4.97 2.00 4.50 1.4 5.6 82 11 8 1.45 138 113
15 C1 2.46 2.00 3.50 2.00 7.40 0.8 6.5 52 22 25 0.88 85 44 73 27
C2 1.78 1.78 6.70 2.00 4.80 0.9 9.0 88 17 11 1.20 115 101
25 C1 2.06 2.00 3.63 2.00 6.90 0.7 6.3 42 19 19 1.01 98 41 70 30
C2 2.55 2.00 5.25 2.00 5.50 1.3 7.2 100 21 16 1.03 98 99
40 C1 2.68 2.00 4.10 2.00 5.20 0.9 5.3 46 24 26 0.84 82 38 41 59
C2 2.00 2.00 3.23 2.00 6.05 1.0 4.9 53 20 29 0.85 82 43
60 C1 1.70 1.70 2.16 2.00 5.35 0.5 3.4 19 35 44 0.56 54 10 14 86
C2 2.30 2.00 2.16 2.00 4.43 1.2 2.4 30 36 33 0.64 61 18
100 C1 0.70 0.70 0.29 0.29 0.47 0.2 0.7 2 53 58 0.34 33 1 1 99
C2 0.64 0.64 0.43 0.43 0.52 0.3 0.8 3 64 53 0.31 30 1
MMS 25 µg/mL C1 3.40 2.00 3.37 2.00 5.70 1.1 4.8 53 55 60 0.32 31 16 17 83
C2 2.53 2.00 4.30 2.00 4.53 1.3 4.9 67 63 64 0.26 25 17

*: x 105cells/mL

Adj. conc.: adjusted cell concentration

SG: suspension growth

Adj. RSG: adjusted relative suspension growth

#: counted on a total number of 96 wells

CE2: cloning efficiency

RCE2: relative cloning efficiency

Adj. RTG: adjusted relative total growth

C1: culture 1

C2: culture 2

0: vehicle control (Water for injections)

MMS: methylmethane sulfonate

(1): expressed as active item

Main experiment without S9 mix, 3-hour treatment: mutagenicity results

Doses µg/mL(1) Adj.
RTG
%
CE2   Wells* MF x 10-6 Ratio MF Mean values of C1 and C2
Adj
RTG %
CE2 MF x 10-6 IMF
0 C1 100 1.03 EW 77 71 73 67 140 1.0    
LC 15 17 12 14 80 1.0
SC 4 8 11 16 52 1.0
C2 100 1.05 EW 74 67 75 74 134 1.0 100 1.04 137
LC 16 23 13 19 98 1.0 LC 89
SC 6 6 8 3 30 1.0 SC 41
1.88 C1 78 0.82 EW 74 75 83 69 149 1.1    
LC 18 12 6 15 87 1.1
SC 5 10 7 12 57 1.1
C2 136 1.23 EW 67 72 72 67 132 1.0 107 1.02 140 3
LC 19 15 17 17 79 0.8 LC 83 none
SC 10 11 8 12 46 1.6 SC 51 11
3.75 C1 74 1.03 EW 79 77 78 73 109 0.8  
LC 11 11 13 13 65 0.8
SC 7 8 5 10 40 0.8
C2 94 1.18 EW 74 73 70 72 120 0.9 84 1.11 115 none
LC 12 15 17 13 68 0.7 LC 66 none
SC 10 8 9 11 44 1.5 SC 42 1
7.5 C1 83 1.12 EW 74 73 78 68 121 0.9  
LC 18 14 11 13 70 0.9
SC 4 11 7 15 45 0.9
C2 113 1.45 EW 70 63 63 71 126 0.9 98 1.28 123 none
LC 12 26 17 12 66 0.7 LC 68 none
SC 15 7 16 14 50 1.7 SC 48 7
15 C1 44 0.88 EW 60 52 68 54 282 2.0  
LC 20 19 14 22 124 1.6
SC 19 26 14 24 138 2.7
C2 101 1.20 EW 54 49 57 48 255 1.9 73 1.04 268 131
LC 25 30 27 22 131 1.3 LC 127 39
SC 17 18 14 30 96 3.2 SC 117 76
25 C1 41 1.01 EW 26 31 29 26 609 4.4  
LC 39 31 41 32 230 2.9
SC 41 39 35 45 266 5.1
C2 99 1.03 EW 35 44 36 31 470 3.5 70 1.02 539 402
LC 40 32 31 32 210 2.2 LC 220 132
SC 25 23 34 36 178 6.0 SC 222 182
40 C1 38 0.84 EW 21 20 17 20 948 6.8  
LC 44 38 54 41 367 4.6
SC 41 49 42 49 379 7.3
C2 43 0.85 EW 18 27 17 20 904 6.7 41 0.85 926 789
LC 50 43 45 53 403 4.1 LC 385 297
SC 43 31 38 38 290 9.8 SC 335 294
60 C1 10 0.56 EW 9 8 13 9 2060 14.7  
LC 55 51 48 50 683 8.6
SC 50 56 56 59 772 14.8
C2 18 0.64 EW 16 15 10 18 1464 10.9 14 0.60 1762 1625
LC 50 50 49 39 526 5.4 LC 604 516
SC 48 49 46 52 554 18.8 SC 663 622
100 C1 1 0.34 EW 22 22 16  - 2290 16.4  
LC 36 46 50  - 895 11.3
SC 47 39 40  - 840 16.1
C2 1 0.31 EW 35 33 39  - 1599 11.9 1 0.33 1945 1808
LC 28 24 32  - 557 5.7 LC 726 637
SC 37 46 28  - 786 26.6 SC 813 772
MMS         25 µg/mL C1 16 0.32 EW 49 53 44 51 1042 7.5  
LC 24 17 27 27 444 5.6
SC 26 27 29 24 504 9.7
C2 17 0.26 EW 57 69 45 61 975 7.3 17 0.29 1009 872
LC 23 17 35 16 524 5.4 LC 484 395
SC 18 11 19 19 371 12.6 SC 438 397

(1): expressed as active item

Adj. RTG: adjusted relative total growth

CE2: cloning efficiency

*: empty wells are counted on a total number of 96 wells

MF: mutation frequency

IMF: Induced Mutation Frequency

 -: not evaluated due to the severe toxicity observed

C1: culture 1

C2: culture 2

0: vehicle control (Water for injections)

MMS: methylmethane sulfonate

EW: empty wells

LC: wells with large colonies

SC: wells with small colonies

Main experiment with S9 mix, 3-hour treatment: cytotoxicity results

Doses µg/mL(1) Post-treat.
Day 0
cell count*
Adj. Conc.* Cell
count*
Day 1
Adj. Conc.* Cell
count*
Day 2
Cell
count
factor
SG Adj.
RSG
%
Empty wells # CE2 RCE2% Adj.
RTG
%
Mean Adj. RTG % Decrease in Adj. RTG %
0 C1 1.93 1.93 8.00 2.00 6.35 1.0 13.2 100 37 23 0.73 100 100 100  
C2 1.82 1.82 7.75 2.00 7.30 1.0 15.5 100 17 29 0.89 100 100
1.88 C1 2.73 2.00 8.00 2.00 7.30 1.4 14.6 157 33 24 0.76 104 164 171 None
C2 3.87 2.00 6.85 2.00 7.80 2.1 13.4 183 18 30 0.87 97 177
3.75 C1 1.46 1.46 5.60 2.00 6.15 0.8 11.8 68 24 30 0.79 109 74 95 5
C2 2.60 2.00 8.40 2.00 6.80 1.4 14.3 131 24 30 0.79 89 117
7.5 C1 2.22 2.00 7.45 2.00 6.00 1.2 11.2 98 23 20 0.94 129 126 149 None
C2 3.77 2.00 9.95 2.00 6.75 2.1 16.8 224 36 28 0.69 77 172
15 C1 2.04 2.00 8.20 2.00 6.10 1.1 12.5 100 28 26 0.79 109 110 110 None
C2 2.70 2.00 7.75 2.00 6.25 1.5 12.1 116 25 24 0.85 96 110
25 C1 2.10 2.00 7.25 2.00 6.80 1.1 12.3 102 25 30 0.78 107 110 104 None
C2 3.20 2.00 8.10 2.00 5.90 1.8 11.9 135 32 36 0.65 73 98
40 C1 3.20 2.00 5.35 2.00 5.55 1.7 7.4 94 18 30 0.87 119 111 90 10
C2 2.93 2.00 4.30 2.00 7.35 1.6 7.9 82 35 23 0.75 84 69
60 C1 2.22 2.00 4.93 2.00 4.80 1.2 5.9 52 33 36 0.64 88 45 38 62
C2 2.46 2.00 4.03 2.00 5.75 1.4 5.8 50 40 41 0.54 60 30
100 C1 2.75 2.00 1.19 1.19 0.74 1.4 0.4 4 31 33 0.69 94 4 4 96
C2 2.63 2.00 1.72 1.72 1.68 1.4 0.8 8 52 44 0.43 49 4
CPA 3 µg/mL C1 2.46 2.00 7.65 2.00 6.15 1.3 11.8 114 33 44 0.57 79 89 78 22
C2 2.70 2.00 6.40 2.00 6.20 1.5 9.9 95 40 31 0.62 70 66

*: x 105cells/mL

Adj. conc.: adjusted cell concentration

SG: suspension growth

Adj. RSG: adjusted relative suspension growth

#: counted on a total number of 96 wells

CE2: cloning efficiency

RCE2: relative cloning efficiency

Adj. RTG: adjusted relative total growth

C1: culture 1

C2: culture 2

0: vehicle control (Water for injections)

CPA: Cyclophosphamide

(1): expressed as active item

Main experiment with S9 mix, 3-hour treatment: mutagenicity results

Doses(1)µg/mL Adj.
RTG
%
CE2   Wells* MF x 10-6 Ratio MF Mean values of C1 and C2
Adj
RTG %
CE2 MF x 10-6 IMF
0 C1 100 0.73 EW 77 75 70 78 170 1.0  
LC 13 12 15 13 102 1.0
SC 7 9 11 5 60 1.0
C2 100 0.89 EW 79 80 82 77 106 1.0 100 0.81 138  
LC 10 10 9 13 65 1.0 LC 84
SC 7 6 5 7 38 1.0 SC 49
1.88 C1 164 0.76 EW 76 77 73 80 150 0.9  
LC 15 14 15 10 100 1.0
SC 5 7 8 6 46 0.8
C2 177 0.87 EW 80 70 81 74 133 1.3 171 0.81 141 4
LC 10 16 10 17 86 1.3 LC 93 9
SC 6 10 5 6 42 1.1 SC 44 none
3.75 C1 74 0.79 EW 77 82 71 75 145 0.9  
LC 11 10 14 7 73 0.7
SC 8 4 11 14 64 1.1
C2 117 0.79 EW 73 74 83 79 137 1.3 95 0.79 141 3
LC 14 13 10 10 82 1.3 LC 78 none
SC 9 9 3 8 50 1.3 SC 57 8
7.5 C1 126 0.94 EW 75 72 60 72 171 1.0  
LC 12 12 30 15 106 1.0
SC 10 12 7 10 57 1.0
C2 172 0.69 EW 83 75 72 74 170 1.6 149 0.81 170 33
LC 6 10 16 12 89 1.4 LC 97 14
SC 7 11 9 10 74 2.0 SC 66 17
15 C1 110 0.79 EW 68 72 39 72 268 1.6  
LC 17 15 37 14 154 1.5
SC 12 9 23 12 99 1.7
C2 110 0.85 EW 77 76 80 75 129 1.2 110 0.82 199 61
LC 13 12 9 15 80 1.2 LC 117 33
SC 7 8 7 7 46 1.2 SC 73 24
25 C1 110 0.78 EW 65 - 63 75 224 1.3  
LC 9 - 20 9 91 0.9
SC 22 - 14 14 122 2.0
C2 98 0.65 EW 68 58 61 67 319 3.0 104 0.72 271 133
LC 15 16 13 14 126 1.9 LC 108 25
SC 14 22 24 16 170 4.5 SC 146 97
40 C1 111 0.87 EW 38 - 37 37 545 3.2  
LC 22 - 26 29 180 1.8
SC 39 - 40 35 291 4.9
C2 69 0.75 EW 40 40 33 44 598 5.7 90 0.81 571 434
LC 33 34 35 25 268 4.1 LC 224 140
SC 30 27 37 35 274 7.3 SC 282 233
60 C1 45 0.64 EW 24 19 19 28 1134 6.7  
LC 41 35 40 24 354 3.5
SC 45 48 55 48 558 9.3
C2 30 0.54 EW 27 32 24 26 1167 11.1 38 0.59 1151 1013
LC 35 34 46 33 451 7.0 LC 403 319
SC 39 36 38 45 491 13.0 SC 525 476
100 C1 4 0.69 EW 9 7 12 5 1787 10.5  
LC 46 39 40 44 422 4.1
SC 64 63 61 62 767 12.8
C2 4 0.43 EW 18 25 22 27 1649 15.6 4 0.56 1718 1580
LC 36 39 34 32 528 8.1 LC 475 392
SC 55 43 50 46 812 21.5 SC 789 741
CPA             3 µg/mL C1 89 0.57 EW 34 38 28 22 1004 5.9  
LC 31 28 29 37 345 3.4
SC 39 35 45 47 496 8.3
C2 66 0.62 EW 31 42 39 31 794 7.5 78 0.60 899 761
LC 28 28 23 38 292 4.5 LC 319 235
SC 44 31 43 35 409 10.8 SC 452 403

0: vehicle control (water for injections)

CPA: Cyclophosphamide

CE2: cloning efficiency

Adj. RTG: adjusted relative total growth

*: empty wells are counted on a total number of 96 wells

EW: empty wells

IMF: Induced Mutation Frequency

(1): expressed as active item

 -: aberant value (0 empty wells)

MF: mutation frequency

C1: culture 1

C2: culture 2

LC: wells with large colonies

SC: wells with small colonies

Conclusions:
Under the experimental conditions of this study, the test item Phosphonium, tetrakis(hydroxymethyl)-, sulphate(2:1); polymer with urea showed a mutagenic activity in the mouse lymphoma assay, both in the presence and absence of a rat liver metabolizing system.
Executive summary:

SUMMARY

The objective of this study was to evaluate the potential of the test item, Phosphonium, tetrakis(hydroxymethyl)-, sulphate(2:1); polymer with urea, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells.

 

Methods

After a preliminary cytotoxicity test, the test item Phosphonium, tetrakis(hydroxymethyl)-, sulphate(2:1); polymer with urea, diluted in water for injections, was tested in a single experiment, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 105cells/mL were exposed to the test or control items for 3 hours, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a 37°C, 5% CO2humidified incubator.

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

 

Results

Since the test item was found to be severely cytotoxic in the preliminary test, the selection of the highest dose level for the main experiment was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.

The Cloning Efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.

The selected dose levels were 1.88, 3.75, 7.5, 15, 25, 40, 60 and 100 µg/mL, both with and without S9 mix.

No precipitate was observed in the culture medium, at any of the tested dose levels, at the end of the treatment period.

 

Cytotoxicity

In the absence of S9 mix, a slight to severe cytotoxicity was induced at dose levels ≥ 40 µg/mL, as shown by a 59 to 99% decrease in the Adj. RTG. The recommended level of cytotoxicity was reached at the dose level of 60 µg/mL (mean Adj. RTG of 14%).

In the presence of S9 mix, a moderate to severe cytotoxicity was induced at dose levels ≥ 60 µg/mL, as shown by a 62 to 96% decrease in the Adj. RTG. None of the selected dose levels induced the recommended level of cytotoxicity. However, considering the narrow dose levels spacing used, this selection was considered as suitable to allow a reliable interpretation.

 

Mutagenicity

Noteworthy increases in the MF exceeding the GEF of 126 x 10-6were noted at dose-levels15 µg/mL in the absence of S9 mix or25 µg/mL in the presence of S9 mix, together with a statistically significant trend (p < 0.0001).

These results met the criteria of a positive response, both with and without S9 mix.

 

Conclusion

Under the experimental conditions of this study, the test item Phosphonium, tetrakis(hydroxymethyl)-, sulphate(2:1); polymer with urea showed a mutagenic activity in the mouse lymphoma assay, both in the presence and absence of a rat liver metabolizing system.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
2016-11-02
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
TK+/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).

MEDIA USED
Cell cultures were grown at 37°C in a humidified atmosphere of 5% CO2/95% air in culture medium. The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium was supplemented by heat inactivated horse serum at 10% (v/v).
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Since the test item was found to be cytotoxic in the preliminary test, the highest dose level selected for the main experiment was based on the level of cytotoxicity, according to the criteria specified in the international regulations:
With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the 3-hour treatment were 3.13, 6.25, 12.5, 18.8, 25, 37.5, 66.7 and 100 µg/mL, with and without S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s): water for injection
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No
- Exposure duration: 3h and 24h without S9-mix and 3h with S9-mix
- Expression time (cells in growth medium): Population doubling

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After the final cell counting, the cells were washed with culture medium containing 10% inactivated horse serum and 1% pluronic acid. The cells were suspended in 49.5% culture medium containing 10% inactivated horse serum, 50% PBS and 0.5% pluronic acid, before being fixed.
Following the fixation, the cells were kept at 4°C for at least an overnight period.

Depending on the observation at the end of the recovery period (presence or absence of precipitate and/or cytotoxicity), at least four dose levels of the test item-treated cultures were selected for spreading on slides. Cells were dropped onto clean glass slides. The slides were air dried before being stained for approximately 15 min in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above


NUMBER OF CELLS EVALUATED: 1000

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995) (e), according to the following criteria:
 micronuclei should be clearly surrounded by a nuclear membrane,
 the micronucleus area should be less than one-third of the area of the main nucleus,
 non-refractility of the micronuclei,
 micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
 micronuclei should be located within the cytoplasma of the cell,
 only mononucleated cells with a number of micronuclei ≤ 5 should be scored to exclude apoptosis and nuclear fragmentation.


DETERMINATION OF CYTOTOXICITY
Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.
Evaluation criteria:
The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
 a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
 for at least one dose level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
 a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met
Statistics:
For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the Chi 2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At the highest dose level of 5000 µg/mL, the pH of the culture medium was approximately 6.5 (7.1 for the vehicle control)
- Effects of osmolality: 318 mOsm/kg H2O (288 mOsm/kg for the vehicle control)
- Precipitation: A precipitate was observed in the culture medium at the end of the treatment period, at dose levels ≥ 2500 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
Following the 3-hour treatment without S9 mix, a slight to severe cytotoxicity was observed at dose levels  20 µg/mL, as shown by a 39 to 100% decrease in the PD.
Following the 24-hour treatment without S9 mix, a severe cytotoxicity was observed at dose levels ≥ 20 µg/mL, as shown by a 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a severe cytotoxicity was observed at dose levels ≥ 100 µg/mL, as shown by a 100% decrease in the PD.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 30 x 10e4

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: population doubling

preliminary toxicity test

Treatment conditions Treatment Cell concentration Post-treatment PD PD as % of control Decrease in PD (%)
used for treatment cell count
( x 104cells/mL) ( x 104cells/mL)
without S9 mix:
3-h treatment
+
24-h recovery
Vehicle control 30 73.5 1.3 100  
Test item (µg/mL)(1)
10   30 83.5 1.5 114 none
100   30 11.9 # # #
500   30 8.0 # # #
1000 P 30 10.7 # # #
2500 P 30 8.8 # # #
5000 P 30 1.9 # # #
without S9 mix:
3-h treatment
+
0-h recovery
Vehicle control 30 108.0 1.8 100  
Test item (µg/mL) (1)
5   30 80.5 1.4 77 23
10   30 70.5 1.2 67 33
20   30 66.0 1.1 62 38
50   30 81.0 1.4 78 22
100   30 1.3 # # #
200   30 1.3 # # #
500   30 6.1 # # #
1000 P 30 5.9 # # #
with S9 mix:
3-h treatment
+
24-h recovery
Vehicle control 30 66.0 1.1 100  
Test item (µg/mL)(1)
10   30 67.0 1.2 102 none
100   30 8.4 # # #
500   30 9.3 # # #
1000 P 30 17.3 # # #
2500 P 30 8.8 # # #
5000 P 30 - - - -

(1): expressed as active item

PD: population doubling

Vehicle control: Water for injections

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

-: not evaluated due to the severe precipitate observed on the slide

P: precipitate was noted in the culture medium at the end of treatment

Main experiment - Short treatment without S9 mix (3-h treatment + 24-h recovery), cytotoxicity

  Cell concentration
used for treatment
( x 10 4 cells/mL)
Culture Post-treatment
cell count
(x 10 4 cells/mL)
Mean PD Mean PD
as %
of control
Decrease in PD (%)
Vehicle control 30 C1 107.0 2.0 100  
C2 141.0
Test item (µg/mL)(1)
3.13 30 C1 128.0 2.0 99 1
C2 116.0
6.25 30 C1 120.0 2.1 103 none
C2 138.0
12.5 30 C1 112.0 1.9 91 9
C2 106.0
18.8 30 C1 83.0 1.7 82 18
C2 110.0
25 30 C1 90.0 1.7 83 17
C2 106.0
37.5 30 C1 74.0 1.3 65 35
C2 77.0
66.7 30 C1 29.0 0.2 11 89 (too cytotoxic)
C2 41.0
100 30 C1 12.5 # # #
C2 13.7
Positive controls
MMC (1 µg/mL ) 30 C1 36.0 0.5 22 78
C2 46.0
COL (0.5 µg/mL) 30 C1 26.0 # # #
C2 16.6

(1): expressed as active item

PD: population doubling

Vehicle control: Water for injections

MMC: Mitomycin C

COL: Colchicine C1: Culture 1 C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

Main experiment - Short treatment without S9 mix (3-h treatment + 24-h recovery), cytogenetic results

Treatment Mean PD
as %
of control
Culture Number
of cells
analysed
Number of cells with n micronuclei Total
micronucleated cells
Frequency of
micronucleated 
Ratio treated/control
n = 1 n = 2 n = 3 n = 4 n = 5 per culture per dose cells (0/00)
Vehicle control 100 C1 1000 3 0 0 0 0 3 3 2    
C2 1000 0 0 0 0 0 0
Test item (µg/mL)(1)
3.13 99 C1                      
C2              
6.25 103 C1                      
C2              
12.5 91 C1                      
C2              
18.8 82 C1 1000 1 0 0 0 0 1 3 2 1.0  
C2 1000 2 0 0 0 0 2
25 83 C1 1000 10 1 0 0 0 11 13 7 4.3 *
C2 1000 2 0 0 0 0 2
37.5 65 C1 1000 14 1 0 0 0 15 36 18 12.0 ***
C2 1000 21 0 0 0 0 21
66.7 11 C1                      
C2              
100 # C1                      
C2              
Positive controls
MMC (1 µg/mL ) 22 C1 1000 15 4 0 0 0 19 127 64 42.3 ***
C2 1000 94 10 4 0 0 108
COL (0.5 µg/mL) # C1 1000 27 7 0 1 0 35 48 24 16.0 ***
C2 1000 10 3 0 0 0 13

(1): expressed as active item

PD: population doubling

Vehicle control: Water for injections

MMC: Mitomycin C

COL: Colchicine

C1: Culture 1 C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

*: p < 0.1 ***: p < 0.001

Main experiment - Treatment with S9 mix (3-h treatment + 24-h recovery), cytotoxicity

Treatment Cell concentration
used for treatment
( x 104cells/mL)
Culture Post-treatment
cell count
(x 104cells/mL)
Mean PD Mean PD
as %
of control
Decrease in PD (%)
Vehicle control 30 C1 103.0 1.5 100  
C2 71.0
Test item (µg/mL)(1)
3.13 30 C1 103.0 1.7 112 none
C2 95.0
6.25 30 C1 104.0 1.5 100 none
C2 70.5
12.5 30 C1 83.5 1.5 101 none
C2 91.5
18.8 30 C1 100.0 1.5 100 0
C2 74.0
25 30 C1 84.5 1.3 86 14
C2 66.0
37.5 30 C1 73.0 1.3 87 13
C2 78.0
66.7 30 C1 44.3 0.6 41 59
C2 49.0
100 30 C1 23.0 # # #
C2 23.4
Positive controls
CPA (6 µg/mL ) 30 C1 51.5 0.7 43 57
C2 43.3

(1): expressed as active item

PD: population doubling

Vehicle control: Water for injections

CPA: Cyclophosphamide

C1: Culture 1 C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

Main experiment - Treatment with S9 mix (3-h treatment + 24-h recovery), cytogenetic results

Treatment Mean PD
as %
of control
Culture Number
of cells
analysed
Number of cells with n micronuclei Total
micronucleated cells
Frequency of
micronucleated
Ratio treated/control
n = 1 n = 2 n = 3 n = 4 n = 5 per culture per dose cells (0/00)    
Vehicle control 100 C1 1000 2 0 0 0 0 2 3 2    
C2 1000 1 0 0 0 0 1    
Test item (µg/mL)(1)
3.13 112 C1                      
C2              
6.25 100 C1                      
C2              
12.5 101 C1                      
C2              
18.8 100 C1                      
C2              
25 86 C1 1000 4 0 0 0 0 4 6 3 2.0  
C2 1000 2 0 0 0 0 2
37.5 87 C1 1000 8 0 0 0 0 8 14 7 4.7 **
C2 1000 6 0 0 0 0 6
66.7 41 C1 1000 59 4 1 0 0 64 87 44 29.0 ***
C2 1000 22 1 0 0 0 23
100 # C1                      
C2              
Positive controls
CPA (6 µg/mL ) 43 C1 1000 20 1 0 0 0 21 57 29 19.0 ***
C2 1000 31 3 2 0 0 36

(1): expressed as active item

PD: population doubling

Vehicle control: Water for injections

CPA: Cyclophosphamide

C1: Culture 1 C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

**: p < 0.01 ***: p < 0.001

Conclusions:
Under the experimental conditions of the study, the test item, Phosphonium, tetrakis(hydroxymethyl)-, sulphate(2:1); polymer with urea, induced chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, both in the presence and absence of a rat liver metabolizing system.
Executive summary:

SUMMARY

The objective of this study was to evaluate the potential of the test item, Phosphonium, tetrakis(hydroxymethyl)-, sulphate(2:1); polymer with urea, to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-.

 

Methods

After a preliminary cytotoxicity test, the test item Phosphonium, tetrakis(hydroxymethyl)-, sulphate(2:1); polymer with urea, diluted in water for injections was tested in a single experiment, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

Without S9 mix

3 h treatment + 24 h recovery

With S9 mix

3 h treatment + 24 h recovery

 

Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

After the final cell counting, the cells were washed and fixed. Then, cells from at least three dose levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). Micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Results

Since the test item was found to be cytotoxic in the preliminary test, the highest dose level selected for the main experiment was based on the level of cytotoxicity, according to the criteria specified in the international regulations.

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the 3-hour treatment were 3.13, 6.25, 12.5, 18.8, 25, 37.5, 66.7 and 100 µg/mL, with and without S9 mix.

No precipitate was observed in the culture medium at any dose levels, at the end of the 3-hour treatment period.

 

Cytotoxicity

A slight to severe cytotoxicity was induced at dose levels ≥ 37.5 µg/mL without S9 mix and ≥ 66.7 µg/mL with S9 mix, as shown by a 35 to 100% decrease in the PD.


Micronucleus analysis

The dose levels selected for the micronucleus analysis were as follows:

18.8, 25 and 37.5 µg/mL without S9 mix, the latter inducing only a 35% decrease in the PD, but the higher dose levels being too cytotoxic,

25, 37.5 and 66.7 µg/mL, the latter inducing the recommended level of cytotoxicity (i.e.59% decrease in the PD).

 

Statistically significant increases in the frequency of micronucleated cells relative to the vehicle control were observed at the dose levels of 25 and 37.5 µg/mL without S9 mix and at 37.5 and 66.7 µg/mL with S9 mix. Despite the dose-response relationship was not demonstrated by the linear regression, the frequencies of micronucleated cells increased together with increasing dose levels. Furthermore, at the dose level of 37.5 µg/mL with and without S9 mix as well as at 66.7 µg/mL with S9 mix, the individual and mean frequencies were above the vehicle control historical range [0.0 - 3.5‰]. Overall, these results were considered to meet the criteria for a positive response, both with and without S9 mix.

Since the results from the 3-hour treatment without S9 mix were considered to meet the criteria for a positive response, the 24-hour treatment without S9 mix was not undertaken.

 

Conclusion 

Under the experimental conditions of the study, the test item, Phosphonium, tetrakis(hydroxymethyl)-, sulphate(2:1); polymer with urea, induced chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, both in the presence and absence of a rat liver metabolizing system.

 

 

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in vivo assays, a mouse micronucleus (OECD 474) as well as an UDS (OECD 486) on rat hepatocyte revealed to be clearly negatives.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study to GLP without any deviations.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
other: Wistar Hanlbm: WIST (SPF)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Animal Breeding Services, CH-4414 Fuellinsdorf and Harlan Winkelmann GmbH, D-33178 Borchen
- Age at study initiation: 6-10 weeks
- Weight at study initiation: 177.2-205.6 g
- Fasting period before study: 6 hours or overnight
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: from 4 October 2004 to 14 December 2004
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: acceptable solubility and non-toxicity to the animals
- Concentration of test material in vehicle: 10 ml/kg bw
Duration of treatment / exposure:
2 and 16 hours
Frequency of treatment:
once
Post exposure period:
not applicable
Remarks:
Doses / Concentrations:
175 and 350 mg/kg
Basis:
actual ingested
based on dry content (65.8%)
No. of animals per sex per dose:
4 (males only)
Control animals:
yes
Positive control(s):
2 hours preparation interval: DMH (N,N'-dimethylhydazinedihydrochloride)
- Justification for choice of positive control(s): validated for this assay
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw / 10 ml/kg bw

16 hours preparation interval: 2-acetylaminofluorene
2 hours preparation interval: DMH (N,N'-dimethylhydazinedihydrochloride)
- Justification for choice of positive control(s): validatd for this assay
- Route of administration: oral gavage
- Doses / concentrations: 100 mg/kg bw / 10 ml/kg bw
Tissues and cell types examined:
primary hepatocytes
Evaluation criteria:
A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points (based on historical data)
A group average between 0 and 5 net grains is considered a marginal response. A dose-related increase in nuclear and net nuclear grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive result with less than 5 net grains.
Statistics:
not required.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
reduction of activity, ruffled fur all animals both doses and treatment times
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100-500 mg/kg
- Solubility: soluble in water at all dose levels
- Clinical signs of toxicity in test animals:
500 mg/kg: Mortality of 1/2 males and 2/2 females. Reduction of spontaneous activity, abdominal position, eyelid closure, ruffled fur, apathy in all animals 6 h post dosing.
350 mg/kg: Mortality of 2/2 females. Reduction of spontaneous activity, abdominal position, ruffled fur, apathy in all animals 6 h post dosing.
250 mg/kg: Reduction of spontaneous activity, abdominal position, ruffled fur, apathy in all animals 6 h post dosing.
100 mg/kg: Reduction of spontaneous activity in all animals 6 h post dosing.

The change in the urine colour of the treated animals could be due to the systemic distribution of the test substance, thus demonstrating its bioavailability.

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test substance at any of the treatment periods or dose groups. The inter-individual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of historical controls.

No dose level of the test substance revealed UDS induction in the hepatocytes of the treated animals compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test substance for 2 hours or 16 hours. Therefore the net grain values obtained after treatment with the test substance were consistently negative. No substantial shift to higher values was obtained in the percentage of cells in repair.

Appropriate reference mutagens [DMHat 40 mg/kg and 2-AAF at 100 mg/kg] were used as positive controls.In vivotreatment withDMHor 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Toxic symptoms in the Main Experiment

2 hour exposure

Toxic reactions

Hours post-treatment 175 / 350 mg/kg bw

 

1 h

2 h

Reduction of spontaneous activity

4/4

4/4

Ruffled fur

4/4

4/4

 

16 hour exposure

Toxic reactions

Hours post-treatment 175 / 350 mg/kg bw

 

1 h

16 h

Reduction of spontaneous activity

4/4

4/4

Ruffled fur

4/4

4/4

Urine colour

No observations

Orange

Viability and number of hepatocytes

Treatment

Period

Animal No.

Viability (%)

Number of isolated cells (x 106)

Deionised water

2 h

1

71

530

2

79

300

4

85

468

175 mg/kg bw Proban STi

2 h

5

73

226

6

74

266

7

71

309

350 mg/kg bw Proban STi

2 h

9

78

312

10

72

281

11

74

289

40 mg/kg bwDMH

2 h

13

70

375

14

75

375

15

73

314

Deionised water

16 h

17

75

225

19

72

241

20

71

249

175 mg/kg bw Proban STi

16 h

21

74

363

22

81

267

23

75

315

350 mg/kg bw Proban STi

16 h

25

72

317

26

70

361

27

81

271

100 mg/kg bw 2-AAF

16 h

29

76

235

30

74

252

31

75

296

 

Mean Nucleus, Cytoplasmic Area and Net Grains

Preparation interval: 2 hours

Test group

Animal No.

Mean Nuclear Grain Count

Mean cytoplasmic Grain Count

Mean Net Nuclear Grain counts

Mean Nuclear Grains of Cells in Repair

% cells in repair

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Deionised water

1

8.4

3.8

14.4

6.0

-6.0

5.5

0.0

0.0

0

2

11.9

7.3

17.1

7.5

-5.2

5.4

8.8

4.9

4

4

13.9

6.8

19.9

9.1

-5.9

6.2

5.3

0.6

3

Mean

11.4

2.8

17.1

2.7

-5.7

0.5

4.7

4.4

2

175 mg/kg bw Proban STi

5

15.9

9.9

27.1

12.7

-11.3

9.8

13.0

9.9

2

6

11.8

5.9

20.5

8.5

-8.7

7.7

7.0

3.5

3

7

24.1

14.2

39.5

20.8

-15.3

14.2

17.5

3.5

2

Mean

17.3

6.3

29.0

9.6

-11.8

3.3

12.5

5.3

2

350 mg/kg bw Proban STi

9

18.3

8.4

28.6

12.4

-10.3

8.7

7.3

1.5

3

10

11.4

7.2

21.6

10.6

-10.3

8.6

10.0

0.0

1

11

16.2

6.5

24.7

8.9

-8.5

7.1

5.7

1.2

3

Mean

15.3

3.6

25.0

3.5

-9.7

1.0

.7

2.2

2

40 mg/kg bwDMH

13

26.5

16.9

22.1

15.4

4.4

11.1

13.4

7.7

45

14

22.8

10.2

20.0

8.9

2.8

11.2

12.2

6.8

46

15

34.0

15.9

20.3

8.0

13.8

13.2

20.0

10.6

70

Mean

27.8

5.7

20.8

1.1

7.0

5.9

15.2

4.2

54

SD=Standard Deviation. SD for each animal is deviation between the analysed cells. SD for means is the deviation between results for the animals in group.

 

Mean Nucleus, Cytoplasmic Area and Net Grains

Preparation interval: 16 hours

Test group

Animal No.

Mean Nuclear Grain Count

Mean cytoplasmic Grain Count

Mean Net Nuclear Grain counts

Mean Nuclear Grains of Cells in Repair

% cells in repair

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Deionised water

17

11.5

5.3

16.4

6.2

-4.9

5.3

6.7

2.9

3

19

17.8

7.4

29.0

11.5

-11.3

9.6

9.5

2.9

4

20

14.2

6.0

21.8

8.1

-7.6

7.3

6.0

0.0

2

Mean

14.4

3.1

22.4

6.3

-8.0

3.2

7.4

1.9

3

175 mg/kg bw Proban STi

21

10.3

4.6

21.0

9.2

-10.7

8.2

0.0

0.0

0

22

16.9

7.1

28.8

11.5

-11.9

9.9

7.0

0.0

1

23

17.8

6.9

30.3

11.2

-12.5

8.8

5.0

0.0

1

Mean

15.0

4.1

26.7

5.0

-11.7

0.9

4.0

3.6

1

350 mg/kg bw Proban STi

25

20.4

9.7

28.6

10.9

-8.1

8.8

11.4

5.6

7

26

18.0

8.4

29.1

13.3

-11.2

10.9

6.4

1.1

5

27

28.2

11.1

36.4

15.1

-8.2

11.3

9.8

6.2

12

Mean

22.2

5.3

31.4

4.4

-9.2

1.7

9.2

2.6

8

100 mg/kg bw 2-AAF

 

29

26.2

10.3

22.5

6.4

3.7

9.5

11.9

7.2

43

30

42.1

18.2

31.2

12.5

10.9

15.0

19.1

12.0

64

31

27.3

9.5

24.0

8.0

3.3

10.3

12.5

6.4

45

Mean

31.9

8.9

25.9

4.6

6.0

4.3

14.5

4.0

51

SD=Standard Deviation. SD for each animal is deviation between the analysed cells. SD for means is the deviation between results for the animals in group.

Historical Control Data (1995-2002)

Test group

Net Grains

Treatment period

Number of animals

Range

Mean

Vehicle controls

1.82 to -13.51

-5.64 +/- 2.27

2, 3 or 16 hours

326

Positive controls (DMH)

0.270 to 47.46

15.69 +/- 9.57

2 or 3 hours

125

Positive controls (2-AAF)

2.60 to 80.18

20.44 +/- 10.98

16 hours

200

 

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions reported, the test substance did not induce DNA damage leading to increased repair synthesis in the hepatocytes of treated rats.
Executive summary:

Proban STi was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. The test substance was formulated in deionised water, which was used as the vehicle control. The test substance was administered orally by gavage at 175 and 350 mg/kg bw (dose volume 10 ml/kg bw). After a treatment period of 2 and 16 hours respectively, the animals were anaesthetised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to3HTdR (methyl-3H-thymidine), which is incorporated if UDS occurs.

The highest dose of the test substance administered in the main experiment (350 mg/kg) was estimated in pre-experiments to be close to the maximum tolerated dose. The main experiment was performed using male rats only, since the males could be dosed higher than females. At a dose of 350 mg/kg of the test substance, the treated females died. However, due to the close proximity of the maximum tolerated dose for the females (250 mg/kg) to that of the males (350 mg/kg) and the limited number of animals assessed, the difference in the toxicity observed in males and females is not considered significant.

The change in the urine colour of the treated animals could be due to the systemic distribution of the test substance, thus demonstrating its bioavailability.

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test substance at any of the treatment periods or dose groups. The inter-individual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of historical controls.

No dose level of the test substance revealed UDS induction in the hepatocytes of the treated animals compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test substance for 2 hours or 16 hours. Therefore the net grain values obtained after treatment with the test substance were consistently negative. No substantial shift to higher values was obtained in the percentage of cells in repair.

Appropriate reference mutagens [DMH at 40 mg/kg and 2 -AAF at 100 mg/kg] were used as positive controls. In vivo treatment with DMH or 2 -AAF revealed distinct increases in the number of nuclear and net grain counts.

It was concluded that under the experimental conditions reported, the test substance did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from supporting substance. Guideline study to GLP.
Justification for type of information:
See attached justification document
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Margate, UK
- Age at study initiation: Phase I: 4-13 weeks; Phase II: 9-10 weeks
- Weight at study initiation: not available
- Fasting period before study: not available
- Housing: 5 per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12:12
Route of administration:
oral: unspecified
Vehicle:
Sterilised deionised water.
Duration of treatment / exposure:
Single exposure
Frequency of treatment:
Once
Post exposure period:
24 and 48 hours
No. of animals per sex per dose:
Male: 200 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 200 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 320 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 320 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): Standard positive control substance
- Route of administration: oral
- Doses / concentrations: 65 mg/kg
Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: MTD in preliminary (Phase I) test

TREATMENT AND SAMPLING TIMES : examination of bone marrow from femur at 24 and 48 hours post dosing

DETAILS OF SLIDE PREPARATION: four smears of marrow per slide. Slides were allowed to air dry and stained with polychrome methylene blue and eosin using an automatic staining machine

METHOD OF ANALYSIS:
Slides were coded and scored blind. for each animal, 2 x 1000 polychromatic erythrocytes were examined for the presence of micronuclei. Extended analysis of a further 2000 polychromatic erythrocytes was conducted for female vehicle control and female treated mice at the 24 hour sampling time.
OTHER:
Slides were examined for evidence of cytotoxicity (alterations in ratio of polychromatic to normochromatic erythrocytes in a sample of 1000 erythrocytes).
Evaluation criteria:
As described by Schmid (1976).
Statistics:
Analysis of variance at 24 and 48 hours, separately for males and females.
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Doses producing toxicity: Males - 200mg/kg Females - 320mg/kg At 200 mg/kg, males showed clinical signs of toxicity including splayed gait and urinary incontinence. In females at 320 mg/kg hunched posture was observed.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 200 mg/kg splayed gait and signs of urinary incontinence were observed
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations:
No biologically significant increases in micronucleus
formation were seen in any animals at any timepoint.

Phase I: Determination of MTD

Group

Test substance

Dose (mg/kg)

Sex

No. deaths/No. dosed

1

ITC 826 Concentrate

500

Male

0/2

2

ITC 826 Concentrate

800

Male

2/2

3

ITC 826 Concentrate

500

Male

Female

1/2

2/5

4

ITC 826 Concentrate

320

Male

Female

3/5

0/5

5

ITC 826 Concentrate

200

Male

0/5

 Phase II: Incidence of micronucleated PEs:

Males:  

Group

Treatment

Dose

Mean incidence of MPE/1000 PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

1.0 +/- 0.9

1.5 +/- 0.9

12

Cyclophosphamide

65 mg/kg

16.3 +/- 5.7 **

 

13

ITC 826 Concentrate

200 mg/kg

0.8 +/- 0.5

0.8 +/- 0.3

PE:   polychromatic erythrocytes

MPE:micronucleated polychromatic erythrocytes

SD:   standard deviation

** statistically significant increase p<0.01

 Females (original count):

Group

Treatment

Dose

Mean incidence of MPE/1000 PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

0.2 +/- 0.5

1.1 +/- 0.7

12

Cyclophosphamide

65 mg/kg

15.9 +/- 8.0 **

 

14

ITC 826 Concentrate

320 mg/kg

1.6 +/- 0.7*

0.9 +/- 0.2

PE:   polychromatic erythrocytes

MPE:micronucleated polychromatic erythrocytes

SD:   standard deviation

** statistically significant increase p<0.01

* statistically significant increase p<0.05

A statistically significant increase in micronuclei in high dose females at 24 hours was considered to be due to
low control values and was therefore considered not to be of toxicological significance. An extended count of a further
2000 polychromatic erythrocytes conducted at the 24 hour timepoint in females did not show any increase in the incidence of micronucleated polychromatic erythrocytes.

Females (additional count) 

Group

Treatment

Dose

Mean incidence of MPE/1000 PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

0.8 +/- 0.8

 

14

ITC 826 Concentrate

320 mg/kg

0.7 +/- 0.5

 

 Combined original and additional counts

Group

Treatment

Dose

Mean incidence of MPE/1000 PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

0.5 +/- 0.5

 

14

ITC 826 Concentrate

320 mg/kg

1.2 +/- 0.3*

 

 All means based on 20 observations (4 counts of 1000 PE per animal)

There was no change in polychromatic/normochromatic ratio.

Percentage of polychromatic erythrocytes:

Males:

Group

Treatment

Dose

Mean percentage of PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

44.1 +/- 3.6

45.6 +/- 4.2

12

Cyclophosphamide

65 mg/kg

41.1 +/- 3.2

 

13

ITC 826 Concentrate

320 mg/kg

44.8 +/- 3.5

45.3 +/- 2.3

PE:   polychromatic erythrocytes

SD:   standard deviation

Females:

Group

Treatment

Dose

Mean percentage of PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

35.9 +/- 9.1

36.0 +/- 4.7

12

Cyclophosphamide

65 mg/kg

31.5 +/- 13.2

 

14

ITC 826 Concentrate

320 mg/kg

37.3 +/- 5.1

37.9 +/- 5.5

PE:   polychromatic erythrocytes

SD:   standard deviation

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the test, ITC 826 Concentrate is not clastogenic in the mouse bone marrow micronucleus test.
Executive summary:

ITC 826 Concentrate was evaluated for its ability to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-1 mice. A single oral dose was given to groups of 5 male mice at a dose level of 200 mg/kg and to groups of 5 female mice at a dose level of 320 mg/kg. In each case the dose level used represented the maximum tolerated dose (MTD) based on patterns of clinical signs and lethalities over a four day observation period . Bone marrow samples were taken 24 and 48 hours after dosing.

No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the control values, were seen in males at the 24 hour sampling time or in either males or females at the 48 hour sampling time. Although a small but statistically significant increase in the incidence of micronucleated polychromatic erythrocytes, compared to the vehicle control, was observed at the 24 hour sampling time in females dosed with ITC Concentrate at 320 mg/kg, this was not reproduced in an extended analysis of a further 2000 polychromatic erythrocytes and is therefore considered of no biological significance.

Comparison of the percentage of polychromatic erythrocytes showed no statistically or biologically significant differences in either sex at either of the sampling times between the vehicle control animals and those treated with ITC 826 Concentrate.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increase in the incidence of micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Under the conditions of the test, ITC 826 Concentrate is not clastogenic in the mouse bone marrow micronucleus test.

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

Mode of Action Analysis / Human Relevance Framework

In spite of the fact that the substance was considered to be positive in vitro, in vivo studies did not confirm these results, that is why the test substance is considered to be non genotoxic.

Additional information

Justification for classification or non-classification

The substance is not classified according to the criteria of the CLP Regulation (EC) N°1272/2008.