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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation studies in bacteria (Bowles & Thompson, 2011), in vitro gene mutation studies in mammalian cells (Stewart, 1981) and In vitro cytogenicity studies (Kirby, 1981) concluded that cyanuric acid and its analogues are not genotoxic under the conditions of the studies.  

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 31 August to 10 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OCSPP 870.5100 - Bacterial Reverse Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
main tests: five concentrations of the test item (50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain
Vehicle / solvent:
- Vehicle used: sterile distilled water
- Justification for choice of vehicle: the test item formed a good, doseable suspension in sterile distilled water at 12.5 mg/ml, therefore, this solvent was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without s9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1) and preincubation (experiment 2)

DURATION
- Preincubation period: 20 minutes at 37°C with shaking at approximately 130 rpm prior to addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar (only in experiment 2)
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine (for S. typhimurium strains) or tryptophan (for E. coli)

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. An indication of a positive result as determined by a statistical analysis of the data as recommended by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test item, Monosodium cyanurate monohydrate, was determined to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item, Monosodium cyanurate monohydrate, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control reference items used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A light, opaque oily test item film was observed under an inverted microscope at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study not to GLP or guideline
Qualifier:
according to
Guideline:
other: Proposed guidelines for registering pesticides in the U.S.; Hazard Evaluation: Humans and domestic animals, Section 163.84-1 (43 FR 37388)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Toxicity testing: S. typhimurium: TA100|Spot test: S. typhimurium: TA1535, TA1537, TA97, TA98 and TA100|Plate incorporation test: S. typhimurium: TA1535, TA1537, TA97, TA98 and TA100
Metabolic activation:
with and without
Metabolic activation system:
S9 preparations from liver of Aroclor 1254-induced male Sprague-Dawley rats and Aroclor 1254-induced male CD-1 mice.
Test concentrations with justification for top dose:
Toxicity testing: 10 mg, 3mg, 1 mg, 0.2 mg, 0.04 mg and 0.01 mg of sample per plate for stock no. 2 and 10 mg per plate for stock no. 1.
Spot test: 25 mg per spot
Plate incorporation test: 10 mg, 3 mg, 1 mg, 0.2 mg, 0.04 mg and 0.01 mg of sample/plate
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene, benzo(a)pyrene, NaNO2, 2-aminoanthracene, 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)







Species / strain:
other: Toxicity testing: S. typhimurium: TA100 Spot test: S. typhimurium: TA1535, TA1537, TA97, TA98 and TA100|Plate incorporation test: S. typhimurium: TA1535, TA1537, TA97, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
With metabolic activation: no cytotoxicity at concentrations ≤ 10 mg of sample per plate. Without metabolic activation: no cytotoxicity at concentrations ≤ 10 mg of sample per plate.

Table 1: Table for gene mutation assay with S9 activation

Concentration (mg/plate)

Revertant Colonies / Plate (mean from 3 separate tests)

TA98

TA100

TA1535

TA1537

10

31.7

231

14.3

7.7

3

47.7

260.3

17.3

5.3

1

31.3

266.7

19.3

8.7

0.2

34.7

257.3

14

9.7

0.04

28

254

15.7

10.7

0.01

40

262.3

21.7

10.3

Positive Control

1003

1863

509

270

Negative Control

35.7

255.7

18.7

12.3

Table 2: Table for gene mutation assay without S9 activation

Concentration (mg/plate)

Revertant Colonies / Plate (mean from 3 separate tests)

TA98

TA100

TA1535

TA1537

10

-

294.7

23.7

10.3

3

-

276.3

24

10.3

1

-

279

21.7

8.7

0.2

-

300.3

23.3

8

0.04

-

279.3

19.7

6.7

0.01

-

296.6

19

7.3

Positive Control

-

534

503

721

Negative Control

-

247.3

24.3

8

- not tested

Conclusions:
Interpretation of results (migrated information):
negative

Cyanuric acid was not mutagenic towards Salmonella typhimurium test strains in plate incorporation or spot tests conducted with or without a rat microsomal activation system. No microbial toxicity was observed with or without microsomal activation.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication
GLP compliance:
no
Type of assay:
bacterial gene mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 preparations from liver of Aroclor 1254-induced male Sprague-Dawley rats and a NADPH generating system.
Test concentrations with justification for top dose:
10 , 40, 200, 1000, 3000 and 10000 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene; Sodium nitrite; benzo(a)pyrene; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)



NUMBER OF REPLICATIONS: triplicate




Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Positive controls validity:
valid

Table 1: Average histidine a revertants/plate for Salmonella microbial strains

Monosodium cyanurate (µg/plate)

TA98

TA100

TA1535

TA1537

Without metabolic activation*

10,000

30 + 12

295 + 23

24 + 5

10 + 2

3,000

23 + 7

276 + 11

24 + 4

10 + 3

1,000

37 + 2

279 + 14

22 + 8

9 + 4

200

20 + 9

300 + 36

23 + 9

9 + 4

40

27 + 5

279 + 33

20 + 9

7 + 2

10

30 + 14

297 + 52

19 + 1

7 + 2

Solvent control

21 + 4

247 + 31

24 + 3

8 + 4

Positive controlb

555

534

503

721

With metabolic activation*

10,000

32 + 7

231 + 23

14 + 8

8 + 6

3,000

48 + 17

260 + 34

17 + 4

5 + 5

1,000

31 + 8

267 + 22

19 + 8

9 + 2

200

35 + 4

257 + 18

14 + 3

10 + 3

40

28 + 5

254 + 51

61 + 3

11 + 4

10

40 + 7

262 + 32

22 + 3

10 + 3

Solvent control

36 + 6

256 + 55

19 + 6

12 + 3

Positive controlb

1003

1863

509

270

a Mean and standard deviation for 3 plates

bValue for sinlge plate

* No response significantly different from controls at p < 0.01

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate from Fischer 334 rats administered Aroclor 1254.
Test concentrations with justification for top dose:
1000 µg/mL directly in media.
Positive controls:
yes
Positive control substance:
other: ethyl methanesulfonate; dimethylnitrosamine.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified

Table 1: Frequency of sister chromatid exchanges (SCEs) in CHO cells in the absence of metabolic activation

Treatment

Sister Chromatid Exchanges Without Metabolic Activation

Cytogeneticista

No. of SCEs

SCEs/cell (mean ± SE)

SCE/Chromosome (mean ± SE)

Negative control

I

461

9.22 ± 0.43

0.479 ± 0.022

II

430

8.60 ± 0.41

0.446 ± 0.022

Ethyl methanesulfonate (10-3 M)

I

2267

45.34 ± 0.95

2.325 ± 0.049

II

1214

24.28 ± 0.7

1.261 ± 0.036

Monosodium cyanurate (µg/ml)

1500b

I

460

9.20 ± 0.43

0.471 ± 0.022

II

334

6.68 ± 0.37

0.348 ± 0.019

750 bc

I

490

9.80 ± 0.44

0.502 ± 0.023

II

300

6.00 ± 0.35

0314 ± 0.018

375b

I

471

9.42 ± 0.43

0.495 ± 0.023

II

324

6.48 ± 0.36

0.339 ± 0.019

188 b

I

400

8.00 ± 0.40

0.401 ± 0.020

II

338

6.76 ± 0.37

0.354 ± 0.019

94 b

I

386

7.72 ± 0.39

0.393 ± 0.020

II

400

8.00 ± 0.40

0.418 ± 0.021

Table 2: Frequency of sister chromatid exchanges (SCEs) in CHO cells in the presence of metabolic activation

Treatment

Sister Chromatid Exchanges with Metabolic Activation

Cytogeneticista

No. of SCEs

SCEs/Cell (mean ± SE)

SCE/Chromosome (mean ± SE)

Negative control

I

529

10.58 ± 0.46

0.555 ± 0.024

II

568

11.36 ± 0.48

0.573 ± 0.024

Dimethylnitrosamine

(10 -3M)

I

1309

26.18 ± 0.72

1.388 ± 0.038

II

1667

33.34 ± 0.82

1.704 ± 0.042

Monosodium cyanurate (µg/ml)

1500b

I

547

10.94 ± 0.47

0.567 ± 0.024

II

729

14.58 ± 0.54

0.745 ± 0.028

750 bc

I

575

10.68 ± 0.46

0.564 ± 0.024

II

534

11.50 ± 0.48

0.588 ± 0.025

375b

I

567

9.52 ± 0.44

0.495 ± 0.023

II

476

11.34 ± 0.48

0.584 ± 0.025

188 b

I

613

12.26 ± 0.50

0.642 ± 0.026

II

612

12.24 ± 0.49

0.629 ± 0.025

94 b

I

550

12.00 ± 0.49

0.632 ± 0.026

II

600

11.00 ± 0.47

0.565 ± 0.024

a Each cytogeneticist analysed 50 cells per treatment

b A crystalline material failed to go completely into solution

c Additional material precipitated from the medium

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro DNA damage and/or repair study
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: Study performed to GLP and guideline
Qualifier:
according to
Guideline:
EU Method B.19 (Sister Chromatid Exchange Assay In Vitro)
GLP compliance:
yes
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
other: Chinese hamster ovary (CHO) cells, ATCC CCL 61, CHO-Kl, proline requiring.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver homogenate preparation (S-9)
Test concentrations with justification for top dose:
93.8, 187.5, 375, 750 and 1500 µg/mL
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: M ethyl methanesulfonate (for test without activation) and M dimethylnitrosamine (for test with activation)
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: M2 metaphase cells were present both with and without metabolic activation at a concentration of 5000 µg/mL or less (test solubility limited).
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Table1 :

Treatment

Sister Chromatid Exchanges Without Metabolic Activation

Cytogeneticista

No. of SCEs

SCEs/Cell b (mean ± SE)

SCE/Chromosome b (mean ± SE)

Negative control

A

461

9.22 ± 0.43

0.479 ± 0.022

B

430

8.60 ± 0.41

0.446 ± 0.022

Monosodium cyanurate (µg/ml)

93.8c

A

386

7.72 ± 0.39

0.393 ± 0.020

B

400

8.00 ± 0.40

0.418 ± 0.021

187.5 c

A

400

8.00 ± 0.40

0.401 ± 0.020

B

338

6.76 ± 0.37

0.354 ± 0.019

375 c

A

471

9.42 ± 0.43

0.495 ± 0.023

B

324

6.48 ± 0.36

0.339 ± 0.019

750 c d

A

490

9.80 ± 0.44

0.502 ± 0.023

B

300

6.00 ± 0.35

0.314 ± 0.018

1500 c d

A

460

9.20 ± 0.43

0.471 ± 0.022

B

334

6.68 ± 0.37

0.348 ± 0.019

Ethyl methanesulfonate (10-3 M)

A

2267

45.34 ± 0.95

2.325 ± 0.049

B

1214

24.28 ± 0.70

1.261 ± 0.036

Table 2:

Treatment

Sister Chromatid Exchanges With Metabolic Activation

Cytogeneticista

No. of SCEs

SCEs/Cell b (mean ± SE)

SCE/Chromosome b (mean ± SE)

Negative control

A

529

10.58 ± 0.46

0.555 ± 0.024

B

568

11.36 ± 0.48

0.573 ± 0.024

Monosodium cyanurate (μg/ml)

93.8 c

A

600

12.00 ± 0.49

0.632 ± 0.026

B

550

11.00 ± 0.47

0.565 ± 0.024

187.5 c

A

613

12.26 ± 0.50

0.642 ± 0.026

B

612

12.24 ± 0.49

0.629 ± 0.025

375 c

A

476

9.52 ± 0.44

0.495 ± 0.023

B

567

11.34 ± 0.48

0.584 ± 0.025

750 c d

A

534

10.68 ± 0.46

0.564 ± 0.024

B

575

11.50 ± 0.48

0.588 ± 0.025

1500 c d

A

547

10.94 ± 0.47

0.567 ± 0.024

B

729

14.58 ± 0.54

0.745 ± 0.028

Ethyl methanesulfonate (10-3 M)

A

1309

26.18 ± 0.72

1..388 ± 0.038

B

1667

33.34 ± 0.82

1.704 ± 0.042

a Each cytogeneticist analyzed 50 cells per sample.

b A one-way ANOVA comparing the SCE frequencies in CHO cells exposed to monosodium cyanurate and to the negative control indicated that the variance between treatment groups was not significantly greater than the variance within treatment groups.

c A crystalline material failed to go into solution during dilution of test article in metabolic mixture at these concentrations.

d Additional material precipitated from the metabolic activation mixture.

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed to GLP and guideline
Qualifier:
equivalent or similar to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
2000, 1750, 1500, 1250, 1000, 750, 500, 250, 100 and 50 µg/mL (100 and 50 µg/mL omitted for activated cultures).
The maximum dose was obtained using a suspension, since 2000 µg/mL exceeded the solubility of the test material. The top dose exhibited a small reduction in suspension growth of 10% for the non-activated cultures and 7% for the S-9 activated cultures.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dilution samples were prepared in the F0P medium or S-9 mix
- Justification for choice of solvent/vehicle: Maximum achievable concentration with water as solvent was 200 µg/mL, at which level no toxicity was observed either in the presence or absence of S-9. Use of suspension in F0P medium or S-9 mix provided maximum dose.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 1.0E05 cells/mL (6mL added to test tube)

DURATION
- Exposure duration: 4-h
- Expression time (cells in growth medium): 24, 48 hours


CLONING:
- At the end of the expression period, the cells were placed in a restrictive medium to allow only the TK-/- cells to grow. The restrictive medium used is cloning medium with either BUdR (50 µg/mL) or TFT (2-4 µg/mL). The cloning medium contains 0.35% Noble agar.
- Cell counts were made for each tube to determine the volume of each cell population which yielded 3E06 cells. This volume was removed and, after centrifugation, resuspended.
- Incubation period/temp: 37 deg C for 10-12 days.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1: Cloning data (without metabolic activation)

Compound conc, (µg/ml)

No of colonies/R.M Plate

     1              2                3

Ave #/plate

No of colonies/V.C Plate

     1              2                 3

Ave #/plate

Mutant frequency

Induced mutant frequency

2000

62

55

71

63

214

264

269

249

0.5

0.1

1750

24

24

36

28

247

238

237

241

0.2

-0.2

1500

58

41

60

53

226

219

235

227

0.5

0.1

1250

52

51

48

50

+

+

243

243

0.4

0.0

1000

49

50

51

50

232

265

246

248

0.4

0.0

750

+

+

+

-

241

259

262

254

+

-

500

45

47

33

42

259

238

+

249

0.3

-0.1

250

46

56

41

48

272

277

272

274

0.3

-0.1

100

27

34

25

29

238

251

234

241

0.2

-0.2

50

+

+

+

-

230

217

212

220

+

-

Solvent 1

40

41

27

36

251

230

239

240

0.3/0.4

-

Solvent 2

53

64

58

58

279

265

287

277

0.4

-

Table 2: Cloning data (with metabolic activation)

Compound conc (µg/ml)

No of colonies/R.M Plate

     1              2              3

Ave #/plate

No of colonies/V.C Plate

     1               2                3

Ave #/plate

Mutant frequency

Induced mutant frequency

2000

+

62

53

58

+

+

243

243

0.5

-0.1

1750

45

48

63

52

+

173

180

177

0.6

0.0

1500

44

52

35

44

222

213

245

227

0.4

-0.2

1250

57

56

57

57

228

222

192

214

0.5

-0.1

1000

+

65

63

64

271

229

264

255

0.5

-0.1

750

52

46

54

51

+

227

191

209

0.5

-0.1

500

55

39

41

45

+

202

225

214

0.4

-0.2

250

60

57

62

60

216

201

203

207

0.6

0.0

Solvent 1

49

57

48

51

213

215

221

216

0.5/0.6

-

Solvent 2

+

+

61

61

188

210

180

193

0.6

-

+ = Culture lost

Conclusions:
The test article cyanuric acid (sodium salt) when tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay in the presence and absence of Aroclor induced rat liver S-9 did not cause a significant increase in the mutant frequencies of treated cultures over that of the solvent controls. Under these test conditions, the test article is considered negative in this assay.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 preparations from liver of Aroclor 1254-induced male Sprague-Dawley rats and a NADPH generating system
Test concentrations with justification for top dose:
10 concentrations without activation and 8 concentrations with activation between 50 - 2000 µg/mL directly in S-9 mix or medium.
Positive controls:
yes
Positive control substance:
other: 7,1,2-dimethylbenz[a]anthracene; ethylmethane sulfonate.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified

Table 1: Results of mouse lymphoma assay without metabolic activation

Concentration (µg/ml)

Mouse Lymphoma Assay Without Metabolic Activation

Total Mutant Clones

Relative Cloning Efficiency a (%)

Relative Total Growth b (%)

Mutation Frequency c (10-6)

Solvent controls

58 + 6

100

100

42

Ethyl methane sulfonate (0.5µl/ml)

437 + 15

38

20

929

Monosodium cyanurate

2000

63 + 8

96

67

58 + 9

1750

28 + 7

93

70

23 + 6

1500

53 + 10

88

67

46 + 9

1250

50 + 2

94

106

41d

1000

50 + 1

96

90

40 + 2

750

Culture lost

98

68

Culture lost

500

42 + 8

96

98

36 + 5

250

48 + 8

106

87

35 + 5

Table 2: Results of mouse lymphoma assay with metabolic activation

Concentration (µg / mL)

Mouse Lymphoma Assay with Metabolic Activation

Total Mutant Clones

Relative Cloning Efficiency a

(%)

Relative

Total Growth b

(%)

Mutation Frequency c (10-6)

Solvent controls

51 + 6

100

96

47

7, 12 -Dimethylbenz[a]-anthracene (5 mg/ml)

293 + 28

63

22

538

Monosodium cyanurate

2000

58 + 6

119

111

48

1750

52 + 10

86

82

63 + 17

1500

44 + 9

111

108

39 + 18

1250

57 + 1

104

103

53 + 5

1000

64 + 1

124

126

52 + 5

750

51 + 4

102

105

48 + 11

500

45 + 9

104

110

37 + 2

250

60 + 3

101

103

58 + 3

a Average viable colonies of treated cultures / average viable colonies of solvent controls × 100

b  Suspension growth treated cultures / average suspension growth controls × 100

c  Number of mutant colonies / number of potential viable colonies

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The results of an in vivo bone marrow cytogenetic assay (chromosome aberration assay, Sharma 1981) showed sodium cyanurate to be non genotoxic.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
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: Study not performed to GLP or guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
no
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Weight at study initiation: 160 - 210g
- Diet: ad libitum
- Water: ad libitum



Route of administration:
oral: gavage
Vehicle:
- Vehicle used: 4% carboxymethyl cellulose
- Lot/batch no: Sigma chemical company, Lot 77C-0334
Duration of treatment / exposure:
24 h and 48 h after treatment
Remarks:
Doses / Concentrations:
1.25, 2.50 and 5.00 g/kg bw
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: Intraperitoneal
- Doses / concentrations:0.275 mg/kg
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES : 24 and 48 h after treatment


DETAILS OF SLIDE PREPARATION: Clean labeled slides were dipped in distilled water and the cell suspensions were dropped onto the slides using Pasteur pipettes. The slides were then passed over a flame from an alcohol lamp and allowed to dry inside a fume hood. Eight slides were made from each animal. Cells on the slides were stained in freshly prepared 7% Giemsa stain (Gurr R66 Giemsa, M/15 Sorensens buffer, pH 6.8) for 10 minutes at room temperature. The stained slides were rinsed in deionized water, air dried and dipped in xylene, and coverslips were mounted with Depex mounting medium.


METHOD OF ANALYSIS: 50 cells were analyzed from each animal in the study. 1000 cells per animal were examined to determine the mitotic index (MI).For each metaphase cell analyzed for chromosomal aberrations, the vernier setting, chromosome number, and the number and types of aberrations recorded. Aberrations were classified, as described by Savage (1975), as chromosome-type or chromatid-type and were further classified as deletionsor exchanges. Cells bearing ten or more aberrations were classified as 'severely damaged cells'.


Evaluation criteria:
numbers and types of structural aberrations , mitotic index
Statistics:
For each treatment group, data on aberrations per cell was checked for conformity to a Poisson distribution. If the data followed a Poisson distribution, the means for each group were subjected to a square root transformation. If the results did not conform to a Poisson ditribution, a square-root transformation was perfomed on the numbers of chromosomal aberrations per cell and means from each group were calculated from the transformed data. Using the transformed data, the Students t-test was performed to compare the frequency of chromosomal aberrations per cell in a treatment group with that of a negative control. Differences were considered statistically significant when p < 0.05.
Sex:
male
Genotoxicity:
negative

Table1: Cytogenetic evaluation of bone marrow cells from male rats exposed to sodium cyanurate by oral gavage: 24 hours

Negative control

Low dose (1.25 g/kg)

Mid dose (2.50 g/kg)

High dose (5.0 g/kg)

Positive control (0.275 mg/kg TEM)

No of animals

5

5

5

5

5

Mitotic index (%)

5.28 ± 0.79

5.74 ± 0.71

7.28 ± 0.94

6.49 ± 0.96

4.39 ± 0.60

No of cells analyzed

250

250

238

239

238

No (%) normal cells

231 (92)

231 (92)

224 (94)

227 (95)

159 (67)

Number (%) abnormal cells

19 (8)

19 (8)

14 (6)

12 (5)

79 (33)

Number of gaps per cell (mean ± SEM)

0.05 ± 0.004

0.07 ± 0.008

0.04 ± 0.003

0.03 ± 0.003

0.10 ± 0.008

Number (%) abnormal cells with:

Chromosome deletions

1 (0.4)

1 (0.4)

1 (0.4)

0

8 (3.4)

Chromosome exchanges

0

0

1 (0.4)

1 (0.4)

4 (1.7)

Chromatid deletions

7 (2.8)

8 (3.2)

7 (2.9)

6 (2.5)

50 (21.0)

Chromatid exchanges

8 (3.2)

13 (5.2)

2 (0.8)

5 (2.1)

32 (13.4)

Aneuploidy

4 (1.6)

2 (0.8)

4 (1.7)

1 (0.4)

5 (2.1)

Polyploidy

0

1 (0.4)

1 (0.4)

0

0

Severe damage

0

0

0

0

3 (1.3)

Types of aberrations per cell:

Overall frequency of aberrations (mean ± SEM)

0.08 ± 0.02

0.08 ± 0.02

0.07 ± 0.02

0.05 ± 0.02

0.89 ± 0.12*

Chromosome deletions

0.004

0.004

0.004

0.000

0.040

Chromosome exchanges

0.000

0.000

0.004

0.004

0.020

Chromatid deletions

0.03

0.03

0.03

0.03

0.50

Chromatid exchanges

0.03

0.03

0.01

0.03

0.20

* Significantly different from control, p <0.05

Table 2: Cytogenetic evaluation of bone marrow cells from male rats exposed to sodium cyanurate by oral gavage: 48 hours

Negative control

Low dose (1.25 g/kg)

Mid dose (2.50 g/kg)

High dose (5.0 g/kg)

Positive control (0.275 mg/kg TEM)

No of animals

5

5

5

5

5

Mitotic index (%)

5.25 ± 0.21

5.31 ± 0.18

4.52 ± 0.18

4.37 ± 0.24

5.52 ± 0.24

No of cells analyzed

239

250

250

226

250

No (%) normal cells

224 (94)

229 (92)

224 (90)

210 (93)

110 (44)

Number (%) abnormal cells

15 (6)

21 (8)

26 (10)

16 (7)

140 (56)

Number of gaps per cell (mean ± SEM)

0.06 ± 0.008

0.05 ± 0.005

0.07 ± 0.005

0.04 ± 0.005

0.13 ± 0.009

Number (%) abnormal cells with:

Chromosome deletions

1 (0.40)

0

0

0

9 (3.6)

Chromosome exchanges

1 (0.4)

0

0

0

2 (0.8)

Chromatid deletions

7 (2.9)

11 (4.4)

10 (4)

4 (1.7)

76 (30.4)

Chromatid exchanges

3 (1.3)

7 (2.8)

15 (6)

7 (3)

64 (25.6)

Aneuploidy

3 (1.3)

3 (1)

0

5 (2.2)

10 (4)

Polyploidy

2 (0.8)

3 (1)

1 (0.40)

0

0

Severe damage

0

0

0

0

25 (10)

Types of aberrations per cell:

Overall frequency of aberrations (mean ± SEM)

0.07 ± 0.02

0.11 ± 0.03

0.12 ± 0.03

0.09 ± 0.02

2.36 ± 0.70*

Chromosome deletions

0.004

0

0

0

0.05

Chromosome exchanges

0.004

0

0

0

0.01

Chromatid deletions

0.03

0.06

0.06

0.02

0.95

Chromatid exchanges

0.01

0.03

0.06

0.04

0.45

Within the range finder, no mortality was observed at any dose tested. Mitotic indices ranged from 1.50 to 6.18 percent and showed no compound related effects. A dose of 5 g/kg was considered the MTD. For the definitive cytogenetic study, at the 24 hour sacrifice no mortality was observed at any dose tested. No compound related effects were observed at the doses tested. The positive control showed an approximately ten fold increase in aberrations compared to the untreated controls. At the 48  hour sacrifice no mortality was observed at any dose tested. No compound related effects were observed. The positive control showed approximately thirty fold increase in aberrations compared to the untreated controls.

Conclusions:
Interpretation of results (migrated information): negative
No mutagenic effects were observed at 24 or 48 hours post dosing, in the bone marrow cells of male rats dosed orally with 1.25, 2.5, or 5.00 g/kg sodium cyanurate.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro gene mutation study in bacteria (Ames test):

In the key study (Bowles & Thompson, 2011) Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item, monosodium cyanurate monohydrate, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control reference items used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A light, opaque oily test item film was observed under an inverted microscope at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

In vitro cytogenicity study in mammalian cells (chromosome aberration test):

In the key study (Stewarts 1981) the effect of monosodium cyanurate on sister chromatid exchange (SCE) frequencies in cultured Chinese hamster ovary (CHO) cells was evaluated. Without metabolic activation CHO cells were exposed to five concentrations ranging from 93.8 to 1500 µg/mL. With metabolic activation, CHO cells were exposed to five concentrations ranging from 93.6 to 1500 µg/mL. Monosodium cyanurate did not induce SCEs in CHO cells with or without metabolic activation. In the supporting literature study (Hammond et al 1985) monosodium cyanurate was incubated with CHO cells at concentration s up to 1500 µg/mL. No significant increases in sister chromatid exchanges were observed.

In vitro gene mutation study in mammalian cells (mouse lymphoma assay):

In the key study (Kirby 1981) cyanuric acid (sodium salt) was tested in the L5178Y TK +/- Mouse Lymphoma Mutagenesis Assay with and without metabolic activation by induced rat liver S-9. The cultures treated without activation were cloned over a range of concentrations which produced from 69% to 113% suspension growth, and the cultures receiving S-9 metabolic activation were cloned over a range of concentrations which produced from 94% to 105% suspension growth. The results showed that the test material did not cause a significant increase in mutant frequency. In the supporting literature study (Hammond et al 1985) monosodium cyanurate did not induce forward mutations at the TK locus of L5178Y mouse lymphoma cells up to a concentration of 2000 µg/mL. The percentage total growth ranged from 67 to 106% (without activation) and 82 to 126% (with activation).

In vivo gene mutation:

In the key study (Sharma 1982) the mutagenic potential of sodium cyanurate was evaluated using the in vivo rat bone marrow cytogenetics assay. The test material was administered to male rats by gavage. In each treatment group five animals each were dosed with 0, 1.25, 2.5 and 5.0 g/kg sodium cyanurate. Chromosomal preparations were made from bone marrow cells following 24 and 48 hours exposure.

The frequency of aberrations per cells was compared with that of the negative control (4% carboxymethyl cellulose). None of the doses tested produced means that were significantly different from the negative control. In the supporting literature study (Hammond et al 1985) rats were administered monosodium cyanurate by gavage at single dosages up to 5000 mg/kg and killed 24 and 48 h after dosing. Bone marrow cells were collected and examined for chromosomal aberrations. At the timepoints examined there was no evidence of monosodium cyanurate-induced chromosomal aberrations.

Justification for classification or non-classification

It is concluded that the available data indicate that cyanuric acid has no genotoxicity and therefore does not warrant classification for mutagenicity under DSD or CLP.