Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

TGMDA (both UVCB and monoconstituent substance) is clearly genotoxic in-vitro, which is not surprising by considering the four glycidyl (epoxy) substituents per molecule and knowing that most of the epoxy compounds use to be genotoxic under in-vitro conditions. In total seven in-vivo studies have been performed in bone-marrow (4 studies), in spermatogonia- and in spermatocyte-forming tissues. The sister chromatid exchange study with bone marrow cells was ambiguous and one of the micronucleus tests was positive. An in vivo Mammalian Alkaline Comet Assay was concluded to be positive for the induction of DNA damage in liver and stomach and an in vivo Transgenic Rodent Mutation Assay was concluded to be negative in the liver and glandular stomach, but elicited a statistically significant positive response in the duodenum at the highest dose of 300/200 mg/kg bw/d.

This positive response occurred at doses exceeding the MTC of 50 mg/kgbw/d identified in repeated dose toxicity studies and therefore is considered to represent site of contact effects and irritaion of epithelia tissues.

Link to relevant study records

Referenceopen allclose all

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
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
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
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:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10th September 2017 to 27th December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
transgenic rodent mutagenicity assay
Specific details on test material used for the study:
Test Substance: 4,4’-Methylenedianiline, oligomeric reaction products with
1-chloro-2,3-epoxypropane
Batch Number: AAF1169300
Supplier: Huntsman
CAS Numbers: 28768-32-3 / 28390-91-2
Correction Factor: None
Expiration Date: 10 April 2018
Appearance: Whitish, yellow-orange translucent viscous liquid
Storage Condition: 2-8ºC, protected from light

The test substance was received on 31 May 2017, at ambient temperature, in good
condition. As instructed by the Study Director, a 50 mg aliquot of test substance
was shipped to the bioanalytical test site (MPI Research) on 04 October 2018. The
remaining bulk test substance was disposed of prior to issuance of the final report,
per Sponsor’s instructions.
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
Fischer F344 Big Blue® homozygous transgenic rats

Rats have been used historically in safety evaluation and genotoxicity studies and
are recommended by regulatory agencies. Because this study was conducted in
accordance with regulatory guidelines, alternatives could not be considered.
The Big Blue® in vivo mutation assay is a Transgenic Rodent (TGR) Mutation
assay described in OECD TG 488 (OECD, 2013). TGR assays in general and the
Big Blue® assay in particular have been reviewed (OECD, 2009 and 2011a) and
identified in OECD TG 488 (OECD, 2011b and 2013) as being appropriate to
investigate in vivo mutagenicity in any tissue of interest. In addition, TGR assays
are also recommended to investigate a potential mutagenic mode of action in the
etiology of rodent tumors.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BioReliance colony housed at Taconic Biosciences, Inc., Hudson, NY
- Age at study initiation: ~ 7 weeks
- Assigned to test groups randomly: yes
- Fasting period before study: N/A
- Housing: Animals were housed in an environmentally
controlled room with continuous recording of room temperatures of 69 - 75°F and
relative humidity of 30 - 70%, with a 12-hour light/12-hour dark cycle. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour.
Animals were group housed during acclimation and following test animal selection,
in polycarbonate cages. Cages and feeders were changed at least once weekly.
Animals were transferred to clean racks at least once every other week.
Environmental enrichment (in the form of Nylabones®) was also provided. Access
to the Animal Facility was dictated by Standard Operating Procedures (SOPs) and
was restricted to authorized personnel only.
- Diet (e.g. ad libitum): All animals received TEKLAD Global Diet #2018C (Certified 18% Protein Rodent
Diet, Envigo, Madison, Wisconsin), in stainless steel rodent feeders, ad libitum from
arrival until termination.
- Water (e.g. ad libitum): Drinking water from an automatic watering system was provided ad libitum from
arrival until termination.
- Acclimation period: The animals were acclimated for 7 days (Groups 1-5) or between 5 and 21 days
(Groups 6 and 7), prior to the first dose administration. Animals were observed
daily for signs of illness or death. Prior to randomization, animals were examined
and approved for use by the Veterinarian.

In life dates:
Animal arrival:
12 September 2017
28 September 2017 (Groups 6 and 7)

Terminal Necropsy date:
19 October 2017
02 November 2017 (Groups 6 and 7)
Route of administration:
oral: gavage
Vehicle:
Vehicle Control: PEG 400
Batch Number: BCBS8557V
Supplier: Sigma-Aldrich
Retest Date: 29 February 2020
Appearance: Clear colorless liquid
Storage Condition: Room temperature
Details on exposure:
Route= Oral

A suitable sized bottle, calibrated to the desired volume with a PTFE stir bar, was
used as the formulation container. The required amount of TGMDA was weighed
and added to the container; approximately 80 or 90% of the required amount of
PEG 400 was also weighed and added. The formulation container was tightly
closed and mixed by shaking and sonication, until the test substance appeared to
have dissolved. The remaining amount of PEG 400 was then added to the
container, until the contents were at the calibration line.
Duration of treatment / exposure:
Dosing was initiated on 19 September 2017 (Groups 1-5) and on 03 October 2017
(Groups 6 and 7). Animals in Groups 1-4 and 6-7 were dosed once daily by oral
gavage, at a dose volume of 10 mL/kg (except as noted above), for up to
28 consecutive days. Animals in Group 5 were dosed by oral gavage on Days 1, 2,
3, 12, 19 and 26 only, at a dose volume of 10 mL/kg. Individual dose volumes
were calculated based on the animal’s most recently recorded body weight.
Formulations for Groups 1-4 and 6-7 were stirred for at least 10 minutes prior to
and throughout dosing. Formulations for Group 5 were mixed by swirling prior to
dosing, and were administered within 2 hours of preparation.
Post exposure period:
Blood Collection 1 hour post dosing for Group 1
Blood Collection 1 and 4 hours post dosing for groups 2-3, 6 and 7
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Decreased to 200 mg/kg bw/d starting on day 9
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Discontinued after 7th dose
Dose / conc.:
10 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Initially, the study consisted of five groups of six male F344 Big Blue® transgenic rats/group
(Main cohort) and four groups of four male F344 wild-type rats/group (TK cohort). Vehicle
control animals (Group 1) received the vehicle (PEG 400) and test substance-treated animals
received TGMDA formulated in the vehicle at initial doses of 100, 300 and 1000 mg/kg/day
(Groups 2, 3 and 4, respectively) once daily by oral gavage, for up to 28 consecutive days.
Positive control animals (Group 5) were exposed by oral gavage to 20 mg/kg/dose of ethyl
nitrosourea (ENU) on Days 1, 2, 3, 12, 19 and 26. The dose volume for all treatments was
10 mL/kg, except as noted below.

Two additional groups were added to the Main and TK cohorts (same number of animals/group
and strain as before) at the 10 and 50 mg/kg/day dose levels (Groups 6 and 7, respectively). The
newly added animals were also dosed once daily by oral gavage, for up to 28 consecutive days, at
10 mL/kg.
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control animals (Group 5) were exposed by oral gavage to 20 mg/kg/dose of ethyl
nitrosourea (ENU) on Days 1, 2, 3, 12, 19 and 26. The dose volume for all treatments was
10 mL/kg, escept if noted otherwise.
Tissues and cell types examined:
Surviving Main cohort animals were sacrificed by CO2 overdose on Day 31 and the liver, duodenum, glandular stomach, testes and cauda
epididymis were collected. Protocol specified
tissues (liver, duodenum and glandular stomach) from at least the first five surviving animals/group
from Group 1 (vehicle control), Group 5 (positive control), and Groups 6, 2 and 3 (TGMDA at 10,
100 and 300/200 mg/kg/day, respectively) were processed for DNA isolation and analysis of cII
mutants, following BioReliance SOPs.
End points evaluated included mortality, clinical signs, body weights, body weight changes, organ
weights (where possible) and mutant frequency data.

Liver, glandular stomach, and duodenum will be collected for somatic tissue mutant
analysis. In addition, testes and cauda epididymis will be collected and held frozen but not
initially analyzed for germ cell mutants. Vas deferens will not be collected due to the low
amount of mature sperm relative to that contained in the cauda epididymis. Should a
somatic tissue be positive, the appropriate follow up tissue for evaluating germ cell
mutation \\ill be the contents of the seminiferous tubules in the testes and not caudal
sperm. Should the Study Director and Sponsor determine that germ cell analysis be
needed, analysis of testis will be added by protocol amendment
Tissues will be weighed, flash frozen in liquid nitrogen and stored at or below -60°C.
Strict adherence to the order of collection is not necessary.
Details of tissue and slide preparation:
Liver, duodenum and glandular stomach samples from at least the first five
surviving animals/group for Groups 1, 2, 3, 5 and 6 were processed for DNA
isolation; if not needed for analysis, tissue samples from the sixth animal were held
in reserve. Testes and cauda epididymis were held in frozen storage, but not
analyzed for mutants. Isolated DNA samples were stored at 2-8°C.
Isolated DNA was processed using Packaging Reaction Mix (PRM), purchased from
New York University, New York, NY. This product is similar to Transpack
manufactured by Agilent, Santa Clara, CA. PRM or Transpack are used to isolate
the recoverable lambda shuttle DNA vectors from the genomic DNA and to package
the lambda shuttle vector DNA into empty phage capsids creating infectious lambda
phage particles. Methods generally followed BioReliance SOPs, based on Agilent
instruction manual titled “λ Select-cII Mutation Detection System for Big Blue®
Rodents” (Agilent, 2015) and Agilent instruction manual titled “Transpack
Packaging Extract for Lambda Transgenic Shuttle Vector Recovery” (Agilent
2009b), unless modified slightly to accommodate the PRM requirements.
Statistics:
Dunnett’s test was conducted on body weight, body weight changes, and organ weight data.
Only Groups 2-5 were compared to the vehicle control; all required statistics were based on
a significance value of p < 0.05.
Key result
Sex:
male
Genotoxicity:
positive
Remarks:
No statistically elevated mutant frequency at the cII gene in liver and glandular stomach, but Test Item elicited a statistically significant positive response in the duodenum at the highest dose level evaluated (300/200 mg/kg/day).
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mortality and In-life Observations
Test item related mortality was noted at the 1000 mg/kg/day dose level, where all animals
(Main and TK cohorts) were terminated in moribund condition. In the Main
cohort, two 50 mg/kg/day animals were terminated early. One TK cohort animal treated at 10 mg/kg/day was found dead on Day 11; this was likely an incidental event, unrelated to the treatment.
During cage side observations, thin appearance, decreased motor activity, hunched
posture, ruffled fur and diarrhea were noted at 1000 mg/kg/day in all animals and the
high incidence resulted in euthanasia of these animals without sample collection.
Hunched posture, diarrhea, ruffled or stained fur and/or decreased motor activity were
also noted at lower doses, except for the 100 mg/kg/day level, but the severity was
generally not indicative of moribundity and with the exception of the diarrhea, the
frequency of observation was sporadic, particularly once the dose was changed from
300 to 200 mg/kg/day for Group 3.
Similar observations, along with stained fur in the urogenital region (at 10, 50 and
1000 mg/kg/day on Day 8, and at 300/200 mg/kg/day), were noted during the weekly hands-on examinations.
Ruffled fur and diarrhea were also reported for the 100 mg/kg/day animals, and diarrhea was also noted in two vehicle control animals. The diarrhea was likely associated with
the properties of the vehicle.

Body weight and body weight gain
Severe weight loss was noted at 1000 mg/kg/day and 300 mg/kg/day after one week of
treatment. Statistically significant differences in body weight changes also occurred at 1000 mg/kg/day and 300 mg/kg/day prompting the removal of Group 4 and the decrease in the
Group 3 dose level to 200 mg/kg/day. The mean weight gain at 300/200 mg/kg/day was statistically significantly different after the dose level was decreased. At 100 mg/kg/day, there were no statistically significant differences in mean body weight or body weight gain versus the vehicle control group.
No concurrent vehicle control group was available for the 10 and 50 mg/kg/day dose
groups. The data was evaluated based upon relative weight gain versus Day 1 but
statistical analysis was not performed. The 10 and 50 mg/kg/day groups started the
treatment period with a mean weight similar to the vehicle control group, but ended the
study (Day 31) with mean weight gain of 12 and 11%, respectively, versus 19% for the
vehicle control. Thus, there was little effect on body weight gain at these doses.
No statistically significant or otherwise notable differences were noted for body weights
or body weight changes for the positive control animals, as compared to the current
vehicle controls.

Bioanalysis Results
Three animals/group were sampled at two time points (1 and 4 hours post-dose), for the
following dose levels: 10, 50, 100 and 200 mg/kg/day;
three vehicle control animals were also sampled at the 1-hour time point post-dose. Of
the 27 samples analysed, most were below the lower limit of quantitation (0.1 ng/mL).
Exposure to the test item was not conclusively established at any dose level below
200 mg/kg/day. A positive concentration of 0.152 ng/mL was confirmed (via sample
reanalysis) for vehicle control animal 3032; a clear cause for this positive result could not
be established, but since the control animals are handled prior to the treated groups for all
activities, including the blood collection where samples were taken from the controls,
processed and stored prior to the treated groups being collected contamination of the
vehicle control samples was unlikely.
Organ weights
Organ weights were collected mainly to provide guidance of sample availability for DNA
extraction, and not for interpretation of toxicity. Only liver and testes were weighed
whole, and therefore, any differences (statistically significant or not) in the other weights
(duodenum, glandular stomach, cauda) cannot be evaluated due to potential variability in
the size of the sample collected.
There were no statistically significant or otherwise important differences in the organ
weights between test item treated groups and the control.

Mutant Frequency Results

A summary of the mutant frequency (MF) data is presented in Text Table 2.

Test item treatment did not cause statistically elevated MF at the cII gene in the
liver or the glandular stomach of Big Blue® male rats. However, a positive response was
noted in duodenum, at the highest dose level tested.
The positive control treatment with ENU produced statistically significant increases in
MF for all tissues tested, demonstrating the utility of the test system to detect and
quantify induced mutants, following exposure to a known direct acting mutagen. The
study design and results obtained met protocol-specified assay acceptance criteria and
were consistent with the study requirements of OECD TG 488 for transgenic rodent
mutation assays.

* = Statistically significant (1-Way ANOVA, Dunnett’s test, p < 0.05)

** = Statistically significant (1-Way ANOVA, p < 0.001)

# = Statistically significant (Kruskal-Wallis, p = 0.009)

MF = Mutant Frequency; SD = Standard Deviation

 Dose Level (mg/kg/d)  Liver Mean [median] MF +- SD (x10-6)  Duodenum Mean MF +- SD (x10 -6)  Glandular Stomach Mean MF +- SD (x10 -6)
 0  48.0 [42.3] _- 29.1  29.4 +- 6.1  30.8 +- 14.4
 10  31.5 +- 13.2  37.6 +- 24.1  34.2 +- 10.3
 100  26.2 +- 6.0  52.0 +- 20.9  28.4 +- 9.8
 300/200  45.9 +_ 14.2  77.2* +- 29.9  32.8 +- 4.8
 Positive Control  389.9 [386.1#] +- 17.6  750.5** +- 85.8  577.0** +- 107.1
Conclusions:
Treatment of Fischer 344 Big Blue® rats at doses of 10, 100 and 300/200 mg/kg/day in PEG 400 did not cause statistically elevated mutant frequency at the cII gene in liver and glandular stomach, but elicited a statistically significant positive
response in the duodenum at the highest dose level evaluated (300/200 mg/kg/day). This positive response occurred at doses exceeding the MTC of 50 mg/kgbw/d identified in repeated dose toxicity studies and therefore is considered to represent site of contact effects and irritaion of epithelia tissues.

There was no test item induced mortality at these dose levels although rats given 1000 mg/kg/day were terminated due to severe clinical signs and weight loss on Day 9.
A cohort of non-transgenic F344 rats was similarly dosed and sampled on Day 28 at 1
and 4 hours after dosing, and the samples were analyzed. Exposure to the test item was not
conclusively established at any dose level below 200 mg/kg/day by this analysis.
Executive summary:

The purpose of this study was to determine the effect of the test substance (4,4’-Methylenedianiline, oligomeric reaction products with 1-chloro-2,3-epoxypropane,) on mutant frequency at thecIIgene in liver, glandular stomach and duodenum from male transgenic Fischer 344 Big Blue®rats. The Big Blue®Assay is a Transgenic Rodent (TGR) mutation assay, described in OECD Test Guideline (TG) 488 (OECD, 2013).

 

Initially, the study consisted of five groups of six male F344 Big Blue®transgenic rats/group (Main cohort) and four groups of four male F344 wild-type rats/group (TK cohort). Vehicle control animals (Group 1) received the vehicle (PEG 400) and test substance-treated animals received TGMDA formulated in the vehicle at initial doses of 100, 300 and 1000 mg/kg/day. Positive control animals (Group 5) were exposed by oral gavage to 20 mg/kg/dose of ethyl

nitrosourea (ENU) on Days 1, 2, 3, 12, 19 and 26.The dose volume for all treatments was 10 mL/kg, except as noted.

There was no test item induced mortality, however, due to excessive toxicity observed at 1000 and 300 mg/kg/day after one week of treatment, dosing for Group 4 was discontinued after the 7th dose, and the animals were euthanized in moribund condition on Day 9 with no samples collected. The dose level for Group 3 was decreased to 200 mg/kg/day starting with Day 9, initially by decreasing the dose volume to 6.7 mL/kg (in order to use the existing formulation); the dose volume returned to 10 mL/kg, beginning in Week 3.

Two additional groups were added to the Main and TK cohorts (same number of animals/group and strain as before) at the 10 and 50 mg/kg/day dose levels (Groups 6 and 7, respectively). The newly added animals were also dosed once daily by oral gavage, for up to 28 consecutive days, at 10 mL/kg.

Surviving TK cohort animals were sacrificed by CO2overdose after completion of the last blood collection on Day 28, and discarded without necropsy. Surviving Main cohort animals were sacrificed by CO2overdose on Day 31 and the liver, duodenum, glandular stomach, testes and cauda epididymis were collected, weighed, flash frozen and stored at -60°C or less. Protocol specified tissues (liver, duodenum and glandular stomach) from at least the first five surviving animals/group from Group 1 (vehicle control), Group 5 (positive control), and Groups 6, 2 and 3 (10, 100 and 300/200 mg/kg/day, respectively) were processed for DNA isolation and analysis ofcIImutants, following BioReliance SOPs.

End points evaluated included mortality, clinical signs, body weights, body weight changes, organ weights (where possible) and mutant frequency data.

Excessive weight loss on Day 8 and observation of severe clinical signs resulted in the termination of the 1000 mg/kg/day group (main and TK cohorts) without sample collection and a reduction in the dose level for the 300 mg/kg/day group to 200 mg/kg/day. The animals that survived to scheduled termination gained weight, although the 300/200 mg/kg/day group mean body weight was below the vehicle control mean, and their weight gain from Day 1 to 31 was statistically significantly below the vehicle control value.

 

Treatment of Fischer 344 Big Blue®rats with4,4’-Methylenedianiline, oligomeric reaction products with 1-chloro-2,3-epoxypropaneat doses of 10, 100 and 300/200 mg/kg/day

in PEG 400 did not cause statistically elevated mutant frequency at thecIIgene in liver and glandular stomach, but elicited a statistically significant positive response in the duodenum at the highest dose level evaluated (300/200 mg/kg/day).

This positive response occurred at doses exceeding the MTC of 50 mg/kgbw/d identified in repeated dose toxicity studies and therefore is considered to represent site of contact effects and irritaion of epithelia tissues.

There was no TGMDA-induced mortality at these dose levels, although rats given 1000 mg/kg/day were terminated on Day 9 due to severe clinical signs and weight loss.

A cohort of non-transgenic F344 rats, similarly dosed and sampled on Day 28 at 1 and 4 hours after dosing did not conclusively demonstrate exposure to 4,4’-Methylenedianiline, oligomeric reaction products with 1-chloro-2,3-epoxypropane at any dose level below 200 mg/kg/day.

The positive control treatment with ENU produced statistically significant increases in mutant frequencies for all tissues tested, demonstrating the utility of the test system to detect and quantify induced mutants, following exposure to a known direct acting mutagen. The study design and results obtained met protocol-specified assay acceptance criteria and were consistent with the study requirements of OECD TG 488 for transgenic rodent mutation assays.

 

 4,4’-Methylenedianiline, oligomeric reaction products with 1-chloro-2,3-epoxypropanewas evaluated as positive for causing a statistically significant increase in mutations at thecIIgene in duodenum of male Big Blue®rats under the conditions of testing.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

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 clear mutagenic potential in-vitro.

Information is available from a range of in-vivo mutagenicity assays: Two studies in male germinal tissues, a “nucleus anomaly” (micronucleus) test in bone-marrow and a recent micronucleus study were clearly negative. A sister chromatid exchange test came out ambiguous and was slightly positive at high dose levels (3000 and 5000 mg/kg bw, oral, gavage). A bone-marrow test in rodents was positive at the limit dose of 5000 mg/kg bw.

In a in vivo Mammalian Alkaline Comet Assay was concluded to be positive for the induction of DNA damage in liver and stomach at dose levels of 1000 mg/kg and 2000 mg/kg.

A transgenic rodent assay showed statistically significant elevated mutant frequency at the cII gene in duodenum at 200/300 mg/kg/d.

The different outcome of in-vitro and in-vivo studies is explained by the presence of epoxide hydrolases in most tissues (both microsomal and cytosolic). This enzyme cleaves epoxide substituents efficiently and thereby detoxifies TGMDA. For example, it has been demonstrated in metabolic studies with another glycidyl substituent containing substance (Bisphenol A glycidyl ether, BADGE) that only the overload of the epoxide hydrolase pathway leads to the formation of a genotoxic metabolite (glycidaldehyde). The overload effect is observed at very high doses of BADGE only. Therefore BADGE is approved by EFSA for food contact and is considered to be non-genotoxic in vivo.


Justification for selection of genetic toxicity endpoint

The studies selected as a key studies are standard tests performed in vivo. The studies are rated Klimisch 1.

Justification for classification or non-classification

There is a clear mutagenic potential in vivo.

The substance showed negative results in an In vivo Micronucleus study and positive in an in vivo Mammalian Alkaline Comet Assay. For this reason an additional test, Big Blue Transgenic Rodent Mutation Assay according to OECD 488, was carried out to clarify mutagenic potential. TGMDA showed positive results in the In vivo Big Blue Transgenic Rodent Mutation Assay and must therefore be classified accordingly.