4,4'-Methylenedianiline, oligomeric reaction products with 1-chloro-2,3-epoxypropane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

The substance is mutagenic in-vitro, which is not surprising considering the epoxy-groups, and knowing that most of the epoxy compounds are mutagenic under in-vitro conditions. However, from the five in-vivo tests performed in bone-marrow and spermatogonial cells one assay was also slightly positive, while four were negative. The substance is negative in all spermatogonial tests, but slightly positive in one SCE mutagenicity tests. In a recent micronucleus test the substance was found to be not mutagenic. In Saccharomyces cerevisiae, MY 720 did not cause gene mutations neither in the presence nor in the absence of activating rat liver enzymes. All together, it is considered that TGMDA has no significant mutagenic potential in-vivo.

Link to relevant study records

Reference

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:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo 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

Qualifier:

according to guideline

Guideline:

other: TSCA, FIFRA and Japanese METI/MHLW guidelines

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system 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

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

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

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.

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)

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Several mutagenicity studies have been conducted in in-vitro test systems which all turned out to be positive, indicating a mutagenic potential in-vitro. In addition different in-vivo mutagenicity assays were performed which gave another picture: five test (spermatogonal assays in male germinal tissue, a nucleus anomaly test in bone-marrow and a recent micronucleus study) were clearly negative, and one sister chromatid exchange test was ambiguous. Furthermore, on the monoconstituent substance one bone-marrow test in rodents and one SCE-assay in rodents were slightly positive at very high dose levels (3000 and 5000 mg/kg body weight, oral, gavage). In both assays, only the highest dose showed a slight positive response, while lower dose levels were negative.

It seems as if the mutagenic effects in somatic cells are restricted to very high exposure levels but not exposure levels below 3000 mg/kg body weight (oral route). It is considered that this is due fast detoxification (hydrolysis of the epoxide) by epoxide hydrolases present in plasma, organs and in mitochondra.

Interestingly, in Saccharomyces cerevisiae, no mutagenic potential was observed, neither in the presence nor in the absence of S9- rat liver enzymes. This shows that the potential for in-vivo mutagenicity in eucaryotes is either very low or completely absent

All together, it was judged as a conservative approach that TGMDA as monoconstituent or UVCB substance is mutagenic in vitro, however not mutagenic in in-vivo based on a weight-of-evidence approach.

Justification for selection of genetic toxicity endpoint
The study selected as a key study is a standard test performed in vivo. The study is rated Klimisch 1.

Justification for classification or non-classification

There is a clear mutagenic potential in vitro. The genotoxic effect is due to the presence of glycidyl ether functions. In vivo, due to the presence of epoxide hydrolases present in the plasma and most (if not all) tissues the substance is readily detoxified by epoxide hydrolysis. The reaction is fast and the detoxifying capacity is high (as shown with other substances in the rat, mice, human and other species). The substance is therefore non-genotoxic in the in vivo studies performed. In some (old) studies performed in vivo there was an equivocal result due to an effect at very high doses (>2000 mg/kg) which exceed the top dose levels of the actual guidelines. It is considered that the effect is due to an overload of the detoxifying capacity. In the overall assessment, the substance is genotoxic in vitro and not genotoxic in vivo.