Hexahydro-1,3,5-trimethyl-1,3,5-triazine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity (in vitro mammalian chromosome aberration test) = Positive, OECD 473, Buskens (2010)

Genetic Toxicity (bacterial reverse mutation assay) = Negative, OECD 471, Verspeek-Rip (2009)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-nov-2009 to 02-feb-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study has been performed according to OECD and/or EC guidelines and according to GLP principles

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral

Details on mammalian cell type (if applicable):

- Type and identity of media: Blood samplesBlood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.Culture mediumCulture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.Lymphocyte culturesWhole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test: Without and with S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 1292 µg/mLWithout S9-mix, 24/48hr exposure; 24/48 hr fixation: 33, 100, 333, 1000 and 1292 µg/mLCytogenetic test:Without and with S9-mix, 3hr exposure; 24 hr fixation: 3, 12 and 18 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: Test substance is stable in DMSO for at least 4 hours at room temperature under normal laboratory light conditions is demonstrated over the concentration range 0.336 to 134 mg/ml based on neat Tryzine

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 0.5 µg/ml for a 3 h exposure period

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumDURATION- Preincubation period: 48 hr- Exposure duration: 3 hr (with and without S9-mix)- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrSPINDLE INHIBITOR (cytogenetic assays): colchicineSTAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: duplicates in two independent experimentsNUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per cultureDETERMINATION OF CYTOTOXICITY - Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cellsOTHER EXAMINATIONS: - Determination of polyploidy: yes- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Key result

Species / strain:

lymphocytes: human periphal blood

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No- Effects of osmolality: No - Precipitation: No precipitation in the exposure medium was observed up to including the highest tested dose level of 1292 µg/mLRANGE-FINDING/SCREENING STUDIES: - Toxicity was observed at dose levels of 33 µg/mL and above in the absence and presence of S9, at all treatment periodsCOMPARISON WITH HISTORICAL CONTROL DATA: - The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.ADDITIONAL INFORMATION ON CYTOTOXICITY:- Appropriate toxicity was reached at the dose levels selected for scoring.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

No effects of Hexahydro-1,3,5-Trimethyl-S-Triazine on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

 

It was noted that Hexahydro-1,3,5-Trimethyl-S-Triazine increased the number of polyploid cells both in the absence and presence of S9-mix in a dose dependent manner. This may indicate that Hexahydro-1,3,5-Trimethyl-S-Triazine has the potential to disturb mitotic processes and cell cycle progression.

 

Conclusions:

Interpretation of results: positive
In the absence of S9-mix, the substance induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations, both when gaps were included and excluded. In the presence of S9-mix, the substance induced a statistically significant, increase in the number of cells with chromosome aberrations at the highest tested cytotoxic concentration, both when gaps were included and excluded. Finally, it is concluded that this test is valid and that the substance is clastogenic in human lymphocytes under the experimental conditions described in this report. The substance may have the potential to disturb mitotic processes and cell cycle progression.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-nov-2009 to 30-nov-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate Main study: TA1535, TA1537 and TA98: Without and with S9-mix: 1, 3, 10, 33, 100 and 333 µg/plateExperiment 2:TA1535, TA1537, TA98 and TA100:Without and with S9-mix: 3, 10, 33, 100, 200 and 333 µg/plateWP2uvrA:Without and with S9-mix: 10, 33, 100, 333, 666 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: Test substance is stable in DMSO for at least 4 hours at room temperature under normal laboratory light conditions is demonstrated over the concentration range 0.336 to 134 mg/ml based on neat Tryzine

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9 mix Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix Migrated to IUCLID6: 60 µg/plate in water for TA1537

Positive control substance:

2-nitrofluorene

Remarks:

Without S9 mix Migrated to IUCLID6: 10 µg/plate in DMSO for TA98

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9 mix Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Exposure duration: 48 hourNUMBER OF REPLICATIONS: - Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.NUMBER OF CELLS EVALUATED: 10E8 per plateDETERMINATION OF CYTOTOXICITY - Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.OTHER EXAMINATIONS: - The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.b) The negative response should be reproducible in at least one independently repeated experiment.A test substance is considered positive if:b) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.b) The increase in the mean number of revertant colonies follows the concentration of test substance (dose-response relationship).

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: No precipitation was observed up to and including the top of 5000 µg/plateRANGE-FINDING/SCREENING STUDIES: - In tester strain TA100, toxicity was observed at dose levels of 333 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 1000 μg/plate and above in the absence and presence of S9-mix. COMPARISON WITH HISTORICAL CONTROL DATA: - The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.ADDITIONAL INFORMATION ON CYTOTOXICITY:TA1535: without and with S9: 200 µg/plate and aboveTA1537: without and with S9: 200 µg/plate and aboveTA98: without and with S9: 200 µg/plate and aboveTA100: without and with S9: 200 µg/plate and aboveWP2uvrA: without and with S9: 666 µg/plate and above

Conclusions:

Interpretation of results: negative
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.It is concluded that this test is valid and that Hexahydro-1,3,5-Trimethyl-S-Tiazine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genetic Toxicity (in vivo mammalian erythrocyte micronucleus test) = Negative, OECD 474, Flanders (2012)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 12 June 2012 and 24 July 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes

Remarks:

Date of GLP inspection: 19 - 21 July 2011 Date of GLP signature: 31 August 2011

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

Sufficient albino Hsd: ICR (CD-1®) strain mice were obtained from Harlan Laboratories UK Ltd., Oxon, UK. At the start of the main test the mice weighed 22 to 30g and were approximately six to ten weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding. Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent Diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

The vehicle was supplied as a 10x concentrated solution by Gibco, as follows:Supplier's identification:Dulbecco’s Phosphate Buffered Saline SolutionSupplier's lot number:938281In-house serial number:V-5241Date received:02 November 2011 Expiry date:01 May 2013Storage conditions:Room temperatureFor the purpose of this study the vehicle control item was freshly prepared as required as a solution at the appropriate concentration in distilled water (Aguettant Ltd batch no. 3008688 01).

Details on exposure:

Groups, each of seven male mice, were dosed once only via the oral route with the test item at 200, 100 or 50 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test item at 200 mg/kg was killed after 48 hours. Two additional groups of male mice were included in the study; one group (seven mice) was dosed via the oral route with the vehicle alone (PBS) and a second group (five mice) was dosed orally with cyclophosphamide. Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test. The vehicle and positive control group animals were killed 24 hours following dosing.Animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

Duration of treatment / exposure:

24 or 48 hours.

Frequency of treatment:

Once only.

Post exposure period:

Not applicable.

Remarks:

Doses / Concentrations:Dose concentrations applied and the basis of quantity used.Basis:other: The maximum tolerated dose of 200 mg/kg was used as the maximum dose, with 100 and 50 mg/kg as the two lower dose levels. The test item was formulated in PBS at 20, 10 and 5 mg/ml and dosed at 10 ml/Kg to achieve actual dose levels.

No. of animals per sex per dose:

Groups, each of seven male mice, were dosed once only via the oral route with the test item at 200, 100 or 50 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test item at 200 mg/kg was killed after 48 hours. Two additional groups of male mice were included in the study; one group (seven mice) was dosed via the oral route with the vehicle alone (PBS) and a second group (five mice) was dosed orally with cyclophosphamide.

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control item was supplied by Acros Organics, as follows:Supplier's identification:CyclophosphamideSupplier's lot number:A0302605In-house serial number:R-5358Date received:26 April 2012Expiry date:26 April 2014Storage conditions:Approximately 4ºC in the darkFor the purpose of this study the positive control item was freshly prepared as required as a solution at 5 mg /ml in distilled water (Aguettant Ltd batch no. 3008688 01) and dosed at 10 ml/kg to give a dose level of 50 mg/kg.

Tissues and cell types examined:

Mammalian erythrocytes.

Details of tissue and slide preparation:

Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each surviving animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

Evaluation criteria:

Slide Evaluation:Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. Where possible, the incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.Interpretation of Results:A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.A positive mutagenic response would be demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to the vehicle control group.If these criteria were not fulfilled, then the test item would be considered non genotoxic under the conditions of the test.A positive response for bone marrow toxicity would be demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Modest decreases in the PCE/NCE ratio, clinical signs, and a premature death.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range-finding Toxicity Test:Three out of the four animals dosed with the test item at and above 300 mg/kg were killed in extremis due to the severity of the clinical signs observed. The clinical signs were as follows: hunched posture, ataxia, ptosis, splayed gait, lethargy, decreased respiration, laboured respiration, pallor of the extremities, pilo-erection, tiptoe gait and hypothermia. In animals dosed with the test item at 160 and 200 mg/kg the following clinical signs were observed: hunched posture, ptosis, pilo-erection and tiptoe gait. Therefore, with adequate evidence of toxicity and at the request of the Sponsor, the maximum dose level in the main test was set at 200 mg/kg, the maximum tolerated dose level, with 100 and 50 mg/kg as the two lower dose levels.The test item showed no marked difference in its toxicity to male or female mice; therefore the main test was performed using male mice only.Micronucleus Test:Mortality Data and Clinical ObservationsOne animal died prematurely in the main test in the 48-hour 200 mg/kg dose group. Clinical signs were observed in animals dosed with the test item at 200 mg/kg, in both the 24 and 48 hour groups, and were hunched posture and ptosis. It was considered that the loss of an animal due to premature death did not affect the integrity of the study, with at least five analysable animals being available per group (sex) as recommended in the OECD guidelines.Evaluation of Bone Marrow SlidesA summary of the results of the micronucleus test is given in attached Table 1. Individual and group mean data are presented in attached Tables 2 to 7.Although not statistically significant, modest decreases in the PCE/NCE ratio were observed in all three of the 24-hour dose groups when compared to the vehicle control group. These decreases, together with the observation of clinical signs and a premature death, were taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.The test item was found not to produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Please see Attached "Tables 1 to 7"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:

Interpretation of results (migrated information): negativeThe test item was considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction. 

The study was perford to assess the potential of the test item to produce damage to chromosos or aneuploidy when administered to mice. The method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Mammalian Erythrocyte Micronucleus Test", Method B12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

Methods. 

A range-finding test was performed to find suitable dose levels of the test item, route of administration, and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. Following discussions with the Sponsor, the micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) of 200 mg/kg and with 100 and 50 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Additional groups of mice were given a single oral dose of phosphate buffered saline (PBS) (7 male mice) or dosed orally with cyclophosphamide (5 male mice), to serve as vehicle and positive controls respectively. Vehicle and positive control animals were killed after 24 hours.

Results. 

One animal died prematurely in the main test in the 48-hour 200 mg/kg dose group. Clinical signs were observed in animals dosed with the test item at 200 mg/kg, in both the 24 and 48‑hour groups, and were hunched posture and ptosis. It was considered that the loss of an animal due to premature death did not affect the integrity of the study, with at least five analysable animals being available per group (sex) as recommended in the OECD guidelines.

Although not statistically significant, modest decreases in the PCE/NCE ratio were observed in all three of the 24-hour dose groups when compared to the vehicle control group. These decreases, together with the observation of clinical signs and a premature death, were taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test item was found not to produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Conclusion. 

The test item was considered to be non-mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

In accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R7a, a mammalian erythrocyte micronucleus test was conducted to investigate whether the positive result in the in vitro mammalian chromosome aberration test could be replicated in vivo. The in vivo study was negative (Flanders, 2012), which is a result seen with other similar triazine molecules where in vitro studies have been positive. The in vivo study doses were in excess of those seen in the in positive in vitro study, and tissue exposure is assumed as the study was conducted by oral gavage, and the substance is favourable for uptake from the G.I. tract. There is no reason to doubt that the study proves that the in vitro result was a false positive.

Additional information

In Vitro Assays

OECD 473 (In Vitro Mammalian Chromosome Aberration Test):

Interpretation of results: positive

In the absence of S9-mix, the substance induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations, both when gaps were included and excluded. In the presence of S9-mix, the substance induced a statistically significant, increase in the number of cells with chromosome aberrations at the highest tested cytotoxic concentration, both when gaps were included and excluded.  Finally, it is concluded that this test is valid and that the substance is clastogenic in human lymphocytes under the experimental conditions described in this report. The substance may have the potential to disturb mitotic processes and cell cycle progression.

OECD 471 (Bacterial Reverse Mutation Assay):

Interpretation of results: negative




In Vivo Assays

OECD 474 (Mammalian Erythrocite Micronucleus Test):

Interpretation of results: negative

There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.  The test item was found not to produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Justification for classification or non-classification

Based on the results of the three genetic toxicity studies conducted on hexahydro-1,3,5 -trimethyl-1,3,5 -triazine, the substance does not meet the criteria for mutagenicity in accordance with Regulation (EC) No 1272/2008.