Cyanuric acid

Endpoint summary

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

Reference

Reference 1

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OCSPP 870.5100 - Bacterial Reverse Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Reference 2

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

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.

Reference 3

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 control^b	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 control^b	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

Reference 4

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
	Cytogeneticist^a	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)
1500^b	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
375^b	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
	Cytogeneticist^a	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)
1500^b	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
375^b	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

Reference 5

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

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: other: Chinese hamster ovary (CHO) cells, ATCC CCL 61, CHO-Kl, proline requiring.

Remarks:

Migrated from field 'Test system'.

Table1 :

Treatment	Sister Chromatid Exchanges Without Metabolic Activation
	Cytogeneticist^a	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)
93.8^c	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
	Cytogeneticist^a	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

Reference 6

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

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.

Reference 7

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	41^d
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

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 or test system 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.