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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 09 January 2012 and 22 February 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
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
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(methacryloylamino)-N,N,N-trimethylpropan-1-aminium 4-(C10-13-sec-alkyl)benzenesulfonate
EC Number:
688-159-8
Cas Number:
1024699-81-7
Molecular formula:
UVCB substance - not available
IUPAC Name:
3-(methacryloylamino)-N,N,N-trimethylpropan-1-aminium 4-(C10-13-sec-alkyl)benzenesulfonate
Constituent 2
Reference substance name:
3-(methacryloylamino)propyl trimethylammonium sec-C10-13-alkylbenzene sulfonate
IUPAC Name:
3-(methacryloylamino)propyl trimethylammonium sec-C10-13-alkylbenzene sulfonate
Constituent 3
Reference substance name:
MAPTA-ABS salt
IUPAC Name:
MAPTA-ABS salt
Details on test material:
Sponsor's identification: 1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-propen-1-yl)amino]-,4-C10-13-sec-alkylbenzenesulfonat es
Description : Amber coloured slightly viscous liquid
Batch number : P1118
Purity : 78.6%
CAS number : 1024699-81-7
Date received : 01 July 2011
Expiry date : 01 June 2012
Storage conditions: Room temperature, in the dark, under nitrogen

The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

Test animals

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 20 to 27g 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:
intraperitoneal
Vehicle:
The vehicle was supplied as a 10x concentrated solution by Gibco, as follows:

Supplier's identification: Dulbecco’s Phosphate Buffered Saline Solution
Supplier's lot number: 938281
In-house serial number: V-5241
Date of preparation: 02 November 2011
Expiry date : 01 May 2013
Storage conditions: Room temperature

For 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 intraperitoneal route with the test item at 120, 60 or 30 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 120 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 intraperitoneal 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.

All 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:
Animals were killed 24 or 48 hours after dosing.
Frequency of treatment:
Groups of mice were dosed once only via the intraperitoneal route
Post exposure period:
All animals were observed for signs of overt toxicity and death approximately one hour after dosing and then once daily as applicable and immediately prior to termination.
Doses / concentrations
Remarks:
Doses / Concentrations:
Groups, each of seven mice were dosed once only via the intraperitoneal route with the test item at 120, 60, or 30 mg/kg.
Basis:
other: The maximum tolerated dose of 120 mg/kg was used as the maximum dose, the substance was formulated in Dulbecco’s Phosphate Buffered Saline Solution at 12, 6 and 3 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 intraperitoneal route with the test item at 120, 60, or 30 mg/kg. One group of male mice from each dose level was killed by cervical dislocation approximately 24 hours following treatment and a second group dosed at 120 mg/kg was killed at approximately 48 hours.
Control animals:
yes, concurrent vehicle
Positive control(s):
Five mice were dosed orally with Cyclophosphamide at a dose of 50 mg/kg. Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test

The positive control item was supplied by Acros Organics, as follows:

Supplier's identification: Cyclophosphamide
Supplier's lot number: A0302605
In-house serial number: R-5119
Date received : 06 June 2011
Expiry date : 05 June 2013
Storage conditions: Approximately 4ºC in the dark

For the purpose of this study the positive control item was freshly prepared as required as a solution at the appropriate concentration in distilled water (Aguettant Ltd batch no. 3008688 01).

Examinations

Tissues and cell types examined:
In mitotic cells in which chromosome damage has been caused by the test item or its metabolites, fragments (centric or acentric) or whole chromosomes tend to lag behind in the anaphase stage of cell division. After telophase a large proportion of the fragments are not included in the nuclei of the daughter cells and hence form a single or multiple micronuclei (Howell-Jolly bodies) in the cytoplasm of these cells. These micronuclei are seen in a wide variety of cell types, but erythrocytes are chosen since micronuclei are easily detected in these cells.

A few hours after the last mitosis is completed, erythrocytes expel their nuclei. Immature erythrocytes, less than 24 hours old, stain blue with May-Grünwald/Giemsa due to the presence of minute fragments of nuclear material in the cytoplasm. This material is mainly ribonucleic acid (RNA), which gradually disappears so that more mature erythrocytes (normochromatic erythrocytes) stain pink with May-Grünwald/Giemsa. The immature blue staining cells are known as polychromatic erythrocytes and mauve stained micronuclei are easily detected in this cell type. If scoring is restricted to polychromatic erythrocytes, all the chromosomal damage detected will have been caused during the final cell cycle of the nucleated precursor cells. Thus by examining polychromatic cells at various periods after administration, the effect of the test item over the previous 30 hours can be monitored.

Any toxic effects of the test item on the immature nucleated cells may lead to a reduction in cell division and cell death. This in turn leads to a reduction in cell volume and to compensate for this, peripheral blood is shunted into the bone marrow. If the ratio of polychromatic to normochromatic erythrocytes is scored and found to be significantly lower than the control value, this is taken as being indicative of cytotoxicity.
Details of tissue and slide preparation:
Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each 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:
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.

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 mutagenic 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
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS

Range-finding Toxicity Test

In animals dosed with the test item at 2000 mg/kg via the oral route, diarrhoea was observed in one of the female animals only. Therefore, with insufficient evidence of toxicity or absorption, the intraperitoneal route was investigated using a dose level of 1000 mg/kg initially. Both animals dosed with the test item at 1000 mg/kg died prematurely. In animals dosed with the test item at 100 to 150 mg/kg the following clinical signs were observed: hunched posture, ataxia, pilo-erection, and ptosis. However, due to the duration of the clinical signs observed at 150 mg/kg, the maximum dose level in the main test was set at 120 mg/kg, the considered maximum tolerated dose level, with 60 and 30 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 Observations

There were no premature deaths seen in any of the dose groups in the main test. Clinical signs were observed in animals dosed with the test item at 120 mg/kg in both the 24 and 48 hour groups, and included hunched posture and ptosis.

Evaluation of Bone Marrow Slides

A 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.

Modest statistically significant decreases in the PCE/NCE ratio were observed in both the 24 and 48-hour 120 mg/kg dose groups, and also the 24-hour 30 mg/kg dose group, when compared to the vehicle control group. Although not statistically significant, a modest decrease was also observed the 24 hour 60 mg/kg dose group. These decreases, together with the observation of clinical signs, 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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The 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 chromosomes or aneuploidy when administered to mice. Thethod 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 perford 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 perford using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) of 120 mg/kg and with 60 and 30 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 intraperitoneal 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. 

There were no premature deaths seen in any of the dose groups in the main test. Clinical signs were observed in animals dosed with the test item at 120 mg/kg in both the 24 and 48‑hour groups, and included hunched posture and ptosis.

Modest statistically significant decreases in the PCE/NCE ratio were observed in both the 24 and 48-hour 120 mg/kg dose groups, and also the 24 -hour 30 mg/kg dose group, when compared to the vehicle control group. Although not statistically significant, a modest decrease was also observed the 24‑hour 60 mg/kg dose group. These decreases, together with the observation of clinical signs, 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.

Conclusion. 

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