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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from 2012-10-08 to 2013-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed under GLP and according to guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: TSCA, FIFRA and Japanese METI/MHLW guidelines
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): 4,4'-Methylenedianiline, oligomeric reaction products with 1-chloro-2,3-epoxypropane
- Physical state: Amber coloured extremely viscous liquid
- Storage condition of test material: approximately 4 °C, in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 6-10 weeks old
- Weight at study initiation: 25-30 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: in groups of up to seven in solid-floor polypropylene cageswith wood-flake bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was freshly prepared as required as an emulsion at the appropriate concentration in arachis oil.
Duration of treatment / exposure:
24 h after dosing of 0, 500, 1000 and 2000 mg/kg test item and 50 mg/kg positive control
48 h after dosing of 2000 mg/kg
Frequency of treatment:
single treatment
Post exposure period:
24 h after dosing of 0, 500, 1000 and 2000 mg/kg test item and 50 mg/kg positive control
48 h after dosing of 2000 mg/kg
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 1000, 2000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
7 male mice per group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): the item known to produce micronuclei under the conditions of the test
- Route of administration: oral (gavage)
- Doses / concentrations: 50 mg/kg / 5 mg/mL

Examinations

Tissues and cell types examined:
bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): single treatment and sampling times: 24 or 48 h following dosing

DETAILS OF SLIDE PREPARATION: 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.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. 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.
Evaluation criteria:
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:
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations. All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part 111 (1989). The data was analysed following a (x+1)^1/2 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
Remarks:
at 2000 mg/kg in both the 24 and 48-hour dose groups, a hunched posture and ptosis were observed; A modest but statistically significant decrease in the PCE/NCE ratio was observed in the 48-hour 2000 mg/kg dose when compared to the vehicle control group.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Solubility: an emulsion was prepared
- Clinical signs of toxicity in test animals: at 2000 mg/kg: hunched posture, ataxia and splayed gait
- Evidence of cytotoxicity in tissue analyzed: no data
- Rationale for exposure: It is recommended in the guideline OECD 474 to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
- Harvest times: no data
- High dose with and without activation: not applicable

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay): there was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erytrocytes in animals dosed with the test item when compared to the vehicle control group.
- Ratio of PCE/NCE (for Micronucleus assay): A modest but statistically significant decrease in the PCE/NCE ratio was observed in the 48-hour 2000 mg/kg dose group when compared to the vehicle control group. This decrease, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.
- Appropriateness of dose levels and route: the selected dose level at 2000 mg/kg of the test item, i.e. maximum recommended dose, produced some evidence of toxicity wenn administered via the oral route.
- Statistical evaluation: see attached document (Summary results)

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:

The study was performed to assess the potential of the test item to produce damage to chromosomes 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. 44012008 of 30 May 2008, the US EPA (OPPTS 870.5395), TSCA and FlFRA guidelines, and be acceptable to the Japanese METIIMHLW guidelines for testing of new chemical substances.

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 consultation with the Sponsor, the micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum recommended dose (MRD) of 2000 mg/kg and with 1000 and 500 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 arachis oil (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.

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 2000 mg/kg in both the 24 and 48-hour dose groups, and included hunched posture and ptosis. A modest but statistically significant decrease in the PCE/NCE ratio was observed in the 48-hour 2000 mg/kg dose group when compared to the vehicle control group. This decrease, together with the observation of clinical signs, was 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 considered to be non-mutagenic under the conditions of the test.