2,4,6-trichloro-1,3,5-triazine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro, Gene mutation: (bacterial reverse mutation assay/Ames test/mouse lymphoma forward mutation assay):
- 2012-0066-DGM: mouse lymphoma L5178 Y cells with and without metabolic activiation, negative for all tested concentrations.
- 94-0200-FGM: S. typhimurium TA97a, TA98, TA100, and TA102, negative for all tested strains either with or without activation.
- Lusby et al., 1979: S. typhimurium TA98, TA100, TA1538, TA 1535, negative for all tested strains either with or without activation.
- 2010-0216-FGM: S. typhimurium TA1535, TA 1537, TA98, TA100 and E. coli, equivocal for all tester strains with and without metabolic activation.
- 2015 -0082 -DGM: S. typhimurium TA1535, TA 1537, TA98, TA100 and E. coli, negative for all tester strains with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2010-07-05 to 2010-09-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from liver induced by Aroclor 1254

Test concentrations with justification for top dose:

C1 0.096 mg / plate
C2 0.048 mg / plate
C3 0.024 mg / plate
C4 0.012 mg / plate
C5 0.006 mg / plate

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

True negative controls:

other: negative control = Acetone (vehicle)

Positive controls:

other: -S9= TA100,1535:Sodium azide; TA98:2-Nitrofluorene; TA1537:9Aminoacridine; E.coli WP2:N-Nitroquinoline 1-oxide; TA98:4Nitro1,2phenylene diamine; TA100:Nitro Furantoin; + S9= TA98,100,1535,1537, E.coli WP2:2-Aminoanthracene; TA 98,100,1537: Benzoapyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min (second experiment)
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: other: Background evaluation

Evaluation criteria:

Tester Strains TA98, TA 100 and WP2uvrA
The test substance is considered positive when, in at least one of these tester strains, it produces at least a 2-fold rise of the increase value over the increase value in the appropriate vehicle control. This rise in the increase value must be accompanied by a positive Dunnet’s test (alpha = 0.05 1-sided) and a dose response when the concentrations of the test substance is increased.

Tester Strains TA1535, TA1537
The test substance is considered positive when, in at least one of these tester strains, it produces at least a 3-fold rise of the increase value over the increase value in the appropriate vehicle control. This rise in the increase value must be accompanied by a positive Dunnet’s test (alpha = 0.05 1-sided) and a dose response when the concentrations of the test substance is increased.

Statistics:

Dunnett´s test (p < 0.05, one sided)

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

A high number of colonies was detected at 48 µg/plate in all tester strains with or without metabolic activation. For the next higher concentration of 98 µg/plate clear signs for cytotoxicity (absent of background) were reported.

It is assumed that the effects observed in this study are due to a phenomenon which was described in a publication of by Mortelmans and Zeiger in 2000. The addition of a small amount of histidine to the top agar allows all the plated bacteria (approximately 1x10^8 cells) to undergo between six and eight cell divisions before the histidine is depleted. However, when a chemical is toxic there may be “thinning” or complete absence of the background lawn compared to the negative or solvent control. Partial toxicity of the chemical will give rise to thinning since not all the plated bacteria were killed or had their growth inhibited. In this case, the surviving bacteria still form microcolonies. Occasionally numerous small non-revertant colonies are present on the plate. The colonies are referred to as “pinpoint colonies” and consist of histidine-dependent bacteria that survived high chemical toxicity. These colonies are readily visible by the naked eye and may be mistaken for revertant colonies. Microscopic inspection of the plates will, however, reveal that there is a total absence of background lawn. The pinpoint colonies arise due to the fact that the high level of toxicity resulted in more histidine being available to the surviving His− bacteria on a per cell basis. Therefore, these bacteria can undergo additional cell divisions until the depletion of the histidine. The histidine dependency can be readily checked by streaking a few pinpoint colonies on GM agar plates supplemented with biotin but without histidine in the absence of the test chemical.

[Mortelmans and Zeiger, 2000]

The verification of the colonies being revertant or non-revertant was not performed in this study. In the study 94-0200-DKM at a concentration 10 times higher than in this assay clearly negative results were obtained. Thus, in conclusion the results of this study remain questionable.

Conclusions:

Interpretation of results (migrated information):
ambiguous without metabolic activation with caution
ambiguous with metabolic activation with caution

An increased number of colonies were found in the concentration range of increasing cytotoxicity. No verification of the colonies being revertant or non-revertant, i.e. if the observation was an artefact due to cytotoxicity, was performed.
Thus, under the conditions of the present bacterial reverse mutation assay the outcome is regarded as equivocal.

Reference 2

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

Study period:

2012-02-24 to 2012-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) locus in L5178Y TK +/- mouse lymphoma cells

Species / strain / cell type:

mammalian cell line, other: L5178Y mouse lymphoma cell line

Details on mammalian cell type (if applicable):

- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes (against the TK -/- phenotyp)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat S9 fraction and an energy producing system comprised of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and glucose-6-phosphate

Test concentrations with justification for top dose:

0, 12.5, 50, 125, 500, 1250 and 2500 µg/mL (preliminary cytotoxicity experiment)
0,1.56, 3.13, 6.25, 12.5 and 25 µg/mL (mutagenicity test)

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Methylmethanesulfonate (without metabolic activation), 3-Methylcholanthrene (with metabolic activation)

Remarks:

Positive controls were not used in the cytotoxicity assay

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h and 24h without S9; 3h with S9 (two independent assays)
- Expression time (cells in growth medium): 2 - 3 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 to 17 days

SELECTION AGENT (mutation assays): 5-trifluoro-thymidine



DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

Evaluation criteria:

Acceptance and evaluation criteria:

- The mutant frequency in the negative control falls within the normal range (historical mean value).
- The plating efficiency (Plating efficiency step 1 and step 2) of the negative control is ≥ 50%.
- At least one concentration of the positive control induces a clear increase in the mutant frequency (the mutant frequency of the positive control is ≥ 2 times the historical mean value of the negative control) with and without S9.
- The mean mutant frequencies (MF) in the negative (vehicle) control cultures fell within the normal range (50 to 170 mutants per 106 viable cells)
- At least one positive control should show either an absolute increase in mean total mutant frequencies (MF) of at least 300 x 10-6 (at least 40% of this should be in the small colony MF), or an increase in small colony mutant frequency of at least 150 x 10-6 above the concurrent vehicle control
- The mean relative Total Growth (RTG) for the positive controls should be greater than 10%
- The mean cloning efficiencies (CE) of the negative controls from the Mutation Experiments were between the range 65% to 120% two days after treatment
- The mean suspension growth (SG) of the negative control replicates from the Mutation Experiments was between the range 8 to 32 following 3-hour treatments or between 32 and 180 following 24-hour treatments
- There should be no excessive heterogeneity between replicate cultures.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

25 µg/mL with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In a preliminary experiment without and with metabolic activation pronounced to complete cytotoxicity (decreased survival) was noted starting at a concentration of 50 µg/mL. Hence, in the main study the concentration-range of 1.56 to 25 µg/mL was used in the experiments without and with metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative controls were consistent with the historical control data.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the conditions of the present mouse lymphoma forward mutation assay in vitro no mutagenicity was observed for Cyanuric chloride with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.05.2016 to 28.07.2016

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

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, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 3; 10; 33; 100; 333; 1000; and 2500 μg/plate

Vehicle / solvent:

acetone

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

sodium azide

Remarks:

Strains: TA 1535, TA 100, Purity: at least 99 % Dissolved in: deionised water: Concentration: 10 μg/plate

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Strains: TA 1537, TA 98 Name: 4-nitro-o-phenylene-diamine, 4-NOPD Purity: > 99.9 % Dissolved in: DMSO (purity >99 %) Concentration: 10 μg/plate in strain TA 98, 50 μg/plate in strain TA 1537

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

Strain: WP2 uvrA Name: methyl methane sulfonate, MMS Purity: > 99.0 % Dissolved in: deionised water Concentration: 2.0 μL/plate

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

Strains: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA Name: 2-aminoanthracene, 2-AA Purity: 97.5 % Dissolved in: DMSO (purity >99 %) Concentration: 2.5 μg/plate (10.0 μg/plate in WP2 uvrA)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Key result

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537, E. coli WP2 uvrA

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:

Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 μg/plate in the absence of S9 mix and from 1000 to 5000 μg/plate in the presence of S9 mix in experiment I and from 1000 to 2500 μg/plate in experiment II.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Cyanuric chloride at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).

Cytotoxicity was observed at the following concentrations:

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500-5000	2500 – 5000	1000 – 2500	2500
TA 1537	2500 – 5000	2500 – 5000	333 – 2500	1000 – 2500
TA 98	2500 – 5000	2500 – 5000	1000 – 2500	1000 – 2500
TA 100	2500 – 5000	2500 – 5000	1000 – 2500	1000 – 2500
WP2 uvrA	5000	5000	2500	2500

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Cyanuric chloride is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 4

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:

July to October 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (e.g. concentration limited to 500 µg/L)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

: TA 1535 not tested, maximum concentration only 500 µg, no cell densities reported

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S. typhimurium TA 97a

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 extract from rat liver (induced with Aroclor 1254)

Test concentrations with justification for top dose:

- 0, 1, 10, 100 and 500 µg/plate
- due to cytotoxicity found at 100 µg/plate and higher doses for TA 97a, dosage was limited to 500 µg/plate for all strains, although no cytotoxicity was reported for any of the other strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: TA 97a: 2-amonifluorene (+ S9) and 4-nitroguineline-N-oxide (- S9), TA98: benzo(a)pyrene (+S9) and 4-nitro-o-phenylene-diamine (-S9), TA100: 2-aminofluorene (+ S9) and sodium azide (-S9), TA 102: 2-aminofluorene (+ S9) and 4-nitroguineline-N-oxide (- S9)

Details on test system and experimental conditions:

- Method of application: in agar (plate incorporation)
- Preincubation period: no preincubation
- Application: 2 mL molten top agar (Difco agar 0.6 %, 0.5 % sodium chloride, 0.05 mM L-histidine HCl and 0.05 mM biotin
0.1 mL of the appropriate test substance or controls
0.1 mL of 10 h grown culture of tester strain
0.5 mL of S9 extract if applicable
are mixed thoroughly and poured onto minimal glucose agar plates(1.5 % Bacto-Difco agar in Vogel and Bonner medium E with 2 % glucose)
- all samples in 4 - 6 replications (positive controls 3 - 5 replications)
- incubation of plates for 2 days at 37 °C
- microscopical analysis of background lawn

- each culture was tested for number of spontaneous revertants, histidine requirement and sensitivity to ampicillin, crystal violet and UV radiation

Evaluation criteria:

two fold or greater increase of revertant colonies above the background of the spontaneous reversion rate

Statistics:

no statistical analysis conducted

Species / strain:

other: TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no significant increase of revertant colonies compared to background revertants

Cytotoxicity / choice of top concentrations:

other: : cytotoxicity reported for concentrations > 100 µg/plate from a pretest, but not significant in the main test

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no significant increase of revertant colonies compared to background revertants

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 500 µg/plate with activation a significant increase of revertants, though lower than a factor of 2 was determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no significant increase of revertant colonies compared to background revertants

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the condition of this bacterial reverse mutation test cyanuric chloride is regarded as not mutagenic.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study with acceptable restrictions (e.g. very limited information on study design, concentrations up to 100 µg/L were tested only). The study was performed with structural analogues (triethylenemelamine (TEM) and 16 other s-triazine compounds).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

- test was performed according to Ames etal, 1975 (Mut. Research, 31, 347-361) with and without activation on strains TA 98, TA 100, TA 1538 and TA 1535

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: TA 98, TA 100, TA 1538 and TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9 aroclor-induced rat liver extract

Test concentrations with justification for top dose:

0 - 100 µg/plate

Species / strain:

other: TA 98, TA 100, TA 1538 and TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

negative results stated for cyanuric chloride

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

17 s-triazine compounds were tested in four salmonella testes strains (TA 98, TA 100, TA 1538 and TA 1535) in present and absence of rat liver extract.
No mutagenicity was observed when similar concentrations of other triazine compounds, including hydroxyatrazine, Aatrex, cyprazine, cyanazine, simazine, 2,4-dioxohexahydro-1 ,3,5-triazine, 2,4-diamino-s-triazine, 2,5,6-tripyridyl-s-triazine, 5-azauracil, 5-azacytosine, ammeline, cyanuric chloride, cyanuric acid, tri-thiocyanuric acid, and s-triazine were tested on all four strains in the presence and absence of the five induced rat liver extracts.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the conditions of the present bacterial reverse mutation assay cyanuric chloride was not mutagenic in four salmonella testes strains (TA 98, TA 100, TA 1538 and TA 1535) in present and absence of rat liver extract.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- two micronucleus tests on bone marrow cells from the rat (OECD TG 475) and an in vivo sister chromatid exchange assay with the rat (EPA OPPTS 870.5915) negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-12-09 to 1986-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: - Guideline study - GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

as at 1983

Deviations:

yes

Remarks:

: in contrast to the OECD guideline from 1997, only 1000 PCE analyzed

Principles of method if other than guideline:

- cells were harvested from bone marrow

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: BOR: NMRI, (SPF Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Versuchstierzucht GmbH & Co. KG., D-4799 Borchen
- Age at study initiation: 6 weeks
- Weight at study initiation: 33 - 39 g (males), 26 - 32 g (females)
- Assigned to test groups randomly: yes, under following basis: computer generated random numbers
- Fasting period before study:
- Housing: 1 per cage (Makrolon cage, type II)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 15 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used:peanut oil
- Justification for choice of solvent/vehicle: non stated, but test substance decomposes in water
- Concentration of test material in vehicle: 61.9 mg/mL
- Amount of vehicle: 10 mL/kg/bw
- Lot/batch no. : not reported
- Purity: not reported
- Identity of vehicle: Peanut oil (Oleum arachidis DAB 8, Fa. H. Lamotte, D-2800 Bremen)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- suspension of the test substance in the vehicle was prepared immediately before administration by adding the test substance to the vehicle. The mixture was homogenized using an ultra turrax homogenizer (Janke u. Kunkel, D-7813 Staufen, Germany)

Duration of treatment / exposure:

- single dosage via gavage

Frequency of treatment:

- single dosage via gavage

Post exposure period:

- 24, 48, and 72 h post administration 6 animals of each sex were sacrificed for examination

Remarks:

Doses / Concentrations:
619 mg/kg bw
Basis:
other: via gavage

No. of animals per sex per dose:

18

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): none stated
- Route of administration: oral via gavage
- Dose: 51.1 mg/kg bw, dissolved in 0.9% saline solution

Tissues and cell types examined:

bone marrow cells from the femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Obvious clinical symptoms of intoxication and deaths were observed at 1000 mg/kg bw in an orientating study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
single dosage via gavage, sampling at 24, 48, and 72 h post administration (6 animals of each sex)

DETAILS OF SLIDE PREPARATION:
- at least two slides per animals were prepared
- animals were sacrificed by CO2 overdose
- both femurs removed and bone marrow cells flushed into labelled centrifuge tubes with 1.5 ml fetal calf serum (Art. No. 210463, Boehringer Mannheim, D-6800 Mannheim 1)
- centrifugation of tubes: 1000 rpm, 5 min (Labofuge 6000, Heraeus-Christ GmbH, D3360 Osterode am Harz)
- supernatant discarded, bone marrow cells suspended upon a thin layer of serum
- the marrow serum suspension was smeared on a slide and dried over night (slide identification: project no., animal no., species, sex, material, date of preparation)
- two slides were stained using the panoptic stain method (Pappenheim et. al, Das Knochenmark, p. 12, ed. Queisser W., Georg Thieme Verlag, Stuttgart, 1978)

METHOD OF ANALYSIS:
- 1000 PCE were scored for the incidence of micronuclei under the microscope (magnification 1000 x, C. Zeiss, D-7082 Oberkochem, Germany) (one slide used per animal, the second as back up)

OTHER:

- The ration of polychromatic to normochromatic erythrocytes was calculated based on 1000 erythrocytes as a measure of the toxic efficacy of the test material

Evaluation criteria:

- If a test material produced no statistical significant and reproducible positive response at any one of the test points compared to the negative control group, it was considered non mutagenic in this system (significance level: 5%)

Statistics:

- The frequencies of micronuclei of the test group and the positive control group were compared with those of the negative control group at each sampling time.
- A POISSON test was applied. Estimation and test for each treatment group and each sex by means of a VAX 750 computer

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

two animals died, one at day 1, one at day 3 post administration

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

two animals died, both on day 2 post administration

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information): negative
Based on the results of the present mammalian erythrocyte micronucleus test cyanuric chloride can be regarded as non mutagenic under the test conditions.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1991-July to 1991-October

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (e.g. no individual animal data)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

as at 1992

Deviations:

no

Principles of method if other than guideline:

Two dose groups (40 and 80 % of the LD50 for ip injection), animals were treated 2 times within 24h, at 6, 24 and 48 h after the last treatment (30, 48 and 72 h after first treatment) 5 animals were sacrificed and bone marrow cells harvested and analyzed.

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Balb/c

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: in-house breeding facility
- Age at study initiation: 8 weeks (males), 9 weeks (females)
- Weight at study initiation: 24 - 26 g (males), 25 - 27 g (females)
- Assigned to test groups randomly: yes, using a computer based random figure generator
- Fasting period before study: 16 h
- Housing: 5 animals per cage (suspended stainless steel wire mesh)
- Diet: standard laboratory diet, ad libitum
- Water: tap water, drinking water quality
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 ± 1.0 °C
- Humidity (%): 50 - 65 %
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: none stated, but the test item is known to decompose in water or other protic solvents
- Concentration of test material in vehicle: not reported
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw
- Purity: not reported

Details on exposure:

- intraperitoneal injections at 0 and 24 hours with cyanuric chloride dissolved freshly in corn oil (3.05 mg/kg bw = 40 % of LD50 and 6.1 mg/kg bw = 80 % of LD50)
- negative controls received corn oil or sterile water

Duration of treatment / exposure:

- 5 animals of each group were sacrificed 30, 48 and 72 respectively after the first administration

Frequency of treatment:

see details on exposure

Post exposure period:

see details on exposure

Remarks:

Doses / Concentrations:
0, 3.05 and 6.10 mg/Kg
Basis:
other: nominal, total dose

No. of animals per sex per dose:

- negative control (the first stage of the experiment): - 5 males
- test material group, 80 % LD50 (the first stage of experiment): - 15 males
- negative control (the second stage of the experiment): - 5 male
- test material group, 40 % L050 (the second stage of the experiment):- 15 males
- test material group, 80 % L.D50 (the second stage of the experiment):- 15 males
- negative control (the second stage of the experiment): - 5 females
- test material group, 80 % LD50 (the second stage of the experiment):- 15 females
- negative control (for positive control compound):- 5 males
- positive control - 15 males

Control animals:

yes, concurrent vehicle

other: yes, sham exposed (= sterile water was injected)

Positive control(s):

- positive controls were treated with 2.5 mg/kg mitomycin C (Sigma, U. K.) dissolved in physilogical saline solution.

Tissues and cell types examined:

- bone marrow cells from the femurs were extracted and analyzed

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- the doses represent 40 and 80% of the LD50 for mice after ip injection

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
see above

DETAILS OF SLIDE PREPARATION:
- both femurs were removed from each mouse and the bone marrow cells flushed out with 1.5 mL fetal calf serum (Serva, Cat. no. 47900), centrifuged at 180 g for 5 min and the supernatant was discarded
- bone marrow cells were suspended in a thin layer of serum of which one drop was smeared on a slide.
- at least four slides per animal were prepared and stained using the panoptic stain method developed by Pappenheim

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes per slide were scored under the microscope for micronuclei

Evaluation criteria:

- If a test material produced no statistical significant and reproducible positive response at any one of the test points compared to the negative control group, it was considered non mutagenic in this system (significance level: 5%)

Statistics:

one way ANOVA

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

10 males in the high dose group died between 6 and 26 h after second administration

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Reduced PCE/NCE ratio at both tested dose indicate a cytotoxic action which is in accordance with an enhanced mortality in the male high dose group (total 10 animal within 24 h post last treatment).

Conclusions:

Interpretation of results (migrated information): negative
Based on the results of the present mammalian erythrocyte micronucleus test cyanuric chloride can be regarded as non mutagenic.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (e.g. publication with very limited information on animal husbandry)

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5915 (In Vivo Sister Chromatid Exchange Assay)

Deviations:

yes

Remarks:

: only males tested

GLP compliance:

no

Type of assay:

sister chromatid exchange assay

Species:

mouse

Strain:

Balb/c

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: in-house breeding facility
- Diet: ad libitum
- Water: ad libitum

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no justification stated, but the test item is hydrophobic and known to degrade in water
- Amount of vehicle: 0.3 mL/animal

Details on exposure:

- 0.3 mL/animal were injected intraperitoneally

Duration of treatment / exposure:

- one single application

Frequency of treatment:

- one single application

Post exposure period:

- not applicable

Remarks:

Doses / Concentrations:
0,8, 16, 24, 32 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- 4 animals
- cyclophosphamid
- 15 mg/kg bw, intraperitoneal

Tissues and cell types examined:

bone marrow from femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- not reported
- in Clayton_1981 a LD50(mice, intraperitoneal) of 10 mg/kg bw was stated

TREATMENT AND SAMPLING TIMES:
- 1 h prior to test item injection animals were lightly anesthesized and tablets of 5-bromodeoxyuridine subcutaneously implanted
- test item or positive/negative control substances were injected intraperitoneal at 0.3 mL/animal
- 21 h post administration bone marrow cell mitosis was inhibited by intraperitoneal administration of colchicine (3.33 mg/kg bw)
- 2 h later animlas were sacrificed and bone marrow of the extracted femurs isolated

DETAILS OF SLIDE PREPARATION:
- flushing of femurs with Hank's solution
- hypotonic treatment of cells with 0.075 M KCl
- fixing of cells with a mixture of 3:1 methanol: glacial acetic acid
- dropping of cells onto chilled slides
- differential staining of sister chromatids (Antoshina, M., M.; Porjadkova, N., A.; "A technique for differential staining of sister chromatids without using fluorochrome." Citol Genet, Vol. 4, p. 349-352, 1978)

METHOD OF ANALYSIS:
- 50 second-generation metaphase cells from each animal were analyzed

Evaluation criteria:

A test substance which does not produce either a statistically significant dose-related increase in the number of SCE or a statistically significant and reproducible positive response at any one of the test points is considered not to induce rearrangements of DNA segments in this system.

Statistics:

- one way ANOVA with Scheffe's test for multiple comparison

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

- table 1, sister chromatid exchange frequency in bone marrow cells:

Dose (mg/kg bw)	No. of animals	SCE/cell ± SD	Mean SCE/cell ± SD
Solvent control (DMSO)	5	4.20 ± 2.36 5.20 ± 2.78 5.32 ± 2.74 4.98 ± 2.06 5.16 ± 2.15	4.97 ± 2.45
8	5	5.06 ± 2.88 5.20 ± 3.32 4.36 ± 1.67 5.20 ± 2.51 4.64 ± 2.00	4.89 ± 2.55
16	5	5.00 ± 2.22 5.36 ± 2.16 5.76 ± 2.34 5.20 ± 2.59 5.32 ± 2.31	5.33 ± 2.33
24	5	5.84 ± 2.84 5.64 ± 2.89 5.32 ± 1.63 5.28 ± 2.03 5.72 ± 2.23	5.56 ± 2.37
32	5	5.08 ± 2.07 5.64 ± 2.27 5.40 ± 2.20 5.16 ± 2.51 5.50 ± 2.60	5.36 ± 2.33
positive control = cyclophosphamide 15 (mg/kg bw)	4	16.44 ± 4.40 15.88 ± 4.26 16.16 ± 4.75 16.16 ± 4.20	16.20 ± 4.18*

* = different from negative control (p 0.025)

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of the present sister chromatid exchange assay in vivo the test substance can be regarded as not genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro:

In a mouse lymphoma forward mutation assay (2012 -0066-DGM) the mutagenic properties of Cyanuric chloride were tested in L5178Y mouse lymphoma cells with and without activation (aroclor induced rat liver S9 extract) in concentrations of 0, 1.56, 3.13, 6.25, 12.5 and 25 µg/mL. Cyanuric chloride was dissolved in acetone and the vehicle served as the negative control. Exposure times were stated with 3h (with and without S9) and 24h (without S9). At the highest concentration tested (25 µg/mL) cytotoxicity was noted in the absence and the presence of metabolic activation. All tested concentrations were within the range of the negative control values and the normal range of 50 to 170 mutants per 106 viable cells. The positive controls Methylmethanesulfonate and 3-Methylcholanthrene caused pronounced increases in the mutation frequencies. Thus, under the conditions of the present study no mutagenicity was observed.

In a bacterial reverse mutation assay (94-0200-DKT) the mutagenic properties of Cyanuric chloride were tested with the S. typhimurium strains TA97a, TA98, TA100, and TA102 with and without activation (aroclor induced rat liver S9 extract) in concentrations of 1, 10, 100 and 500 µg/plate. Thereby, Cyanuric chloride showed no mutagenic properties under the test conditions.
A second bacterial mutation assay (2010-0216-FGM) showed increases of the number of colonies in the S.typhimurium (TA 1535, TA 1537, TA 98 and TA 100) as well as E.coli tester strains at the concentration level of 48 µg/plate, associated with starting cytotoxicity. The response showed no dose-response relationship and was obtained at a concentration 10 times lower than the highest concentration tested negative in the key study 94-0200-DKT. Verification if the colonies were revertants or non-revertants was not performed. Thus, the results are considered equivocal.

In a further recent GLP- and guideline-compliant Ames test (2015 -0082 -DGM), Cyanuric chloride did also not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. For this test the S. typhimurium strains TA98, TA100, TA1535 and TA1537, as well as E. coli WP2 uvrA were used with and without metabolic activation (aroclor induced rat liver S9 extract) at doses of 3, 10, 33 ,100, 333, 1000, 2500, and 5000 μg/plate.

Thus, from the entirety of data on bacterial mutagenicity it can be concluded that the test item does not have mutagenic potential.

In vivo:

In an in vivo mammalian erythrocyte micronucleus test (87-0021-DGM) mice were tested once orally with 619 mg/kg bw Cyanuric chloride in peanut oil. Animals were sacrificed by CO2 inhalation 24, 48 and 72 h post treatment and bone marrow cells were harvested and analyzed for formation of micronuclei in compliance to the guideline. In one animal at the first sampling time a strongly increased number of micronuclei were seen. However in all other animals at this and the other sampling times no differences compared to controls were seen. Therefore the effect in this animal is considered to have occurred by chance. Based on these results Cyanuric chloride can be regarded as non mutagenic under the test conditions.

In a second mammalian erythrocyte micronucleus test (95-0179-FGM) mice were treated 2 times within 24h (3.05 and 6.1 mg/kg). At 6, 24 and 48 h after the last treatment (30, 48 and 72 h after first treatment) 5 animals were sacrificed respectively and bone marrow cells harvested and analyzed for formation of micronuclei in compliance to the guideline. Based on these results Cyanuric chloride can be regarded as non mutagenic under the test conditions.

In a sister chromatid exchange in vivo (Wyszynska etal_1994) mice (5 per dose) were intraperitoneal administered with 0, 8, 16, 24, 32 mg/kg bw Cyanuric chloride. The positive control was Cyclophosamide (15 mg/kg bw). Under the conditions of the present sister chromatid exchange assay in vivo Cyanuric chloride was classified as not genotoxic.

 

Justification for selection of genetic toxicity endpoint
The most reliable studies were selected.

Short description of key information:
Genetic toxicity in vitro, Gene mutation: (bacterial reverse mutation assay/Ames test/mouse lymphoma forward mutation assay):
- 2012-0066-DGM: mouse lymphoma L5178 Y cells with and without metabolic activiation, negative for all tested concentrations.
- 94-0200-FGM: S. typhimurium TA97a, TA98, TA100, and TA102, negative for all tested strains either with or without activation.
- Lusby et al., 1979: S. typhimurium TA98, TA100, TA1538, TA 1535, negative for all tested strains either with or without activation.
- 2010-0216-FGM: S. typhimurium TA1535, TA 1537, TA98, TA100 and E. coli, equivocal for all tester strains with and without metabolic activation.
- 2015 -0082 -DGM: S. typhimurium TA1535, TA 1537, TA98, TA100 and E. coli, negative for all tester strains with and without metabolic activation.
Genetic toxicity in vivo: two micronucleus tests on bone marrow cells from the rat (OECD TG 475) and an in vivo sister chromatid exchange assay with the rat (EPA OPPTS 870.5915) negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Taking into account the overall data for genetic toxicity Cyanuric chloride should not be subjected to classification for mutagenicity according to EU Classifiaction, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008 due to the negative results found in the vitro and in vivo studies available.