Registration Dossier

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Sponsor's identification:Hexahydro-1,3,5-trimethyl-s-triazineDescription:Pale yellow liquidDate received:26 April 2012Storage conditions:Room temperature, in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals 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.

Administration / exposure

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.
Doses / concentrations
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.

Examinations

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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.