p-phenylenebis(methylamine)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames:

One study in 1998 shows negative to S. typhimurium (TA98, 100, 1535, 1537) and E. coli (WP2) strains.

Another study in 1997 shows positive to S. typhimurium TA104, and negative to other S. typhimurium (TA98, 100, 102, 1535, 1537) and E. coli (WP2uvrA, pKM101) strains.

In vitro chromosome aberration:

One study in 2000 shows negative result to CHL cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 1998-04-14 to 1998-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 99.67%

Target gene:

Salmonella typhimurium: histidine
Escherichia coli: tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose-finding study: 10, 50, 100, 500, 1000 and 5000 μg/plate
Main study: 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For all strains with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For strain TA98 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

For strain TA100 and WP2 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For strain TA1535 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For strain TA1537 without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 1E+09 cells/mL

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

The test result are reported positive if the (mean) revertant colony count on the plate treated with the test substance is twice or more the (mean) spontaneous revertant colony count determined in the negative control group and apparent dose-dependence is found between the dose of test substance and the revertant colony count. The test result is reported negative in all other cases.

Key result

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results obtained in the present study indicated that the test substance shows no mutagenic potential since the revertant colony counts determined for each tester strain in both the absence and presence of S9 mix were less than twice the spontaneous revertant colony count determined in the negative control group.
The test substance showed antibacterial activity against TA100, TA98, TA1535 and TA1537 at 2500 μg/plate and higher concentrations in both the presence and absence of S9 mix and against WP2 at 5000 μg/plate in the absence of S9 mix and 1250 μg/plate and higher concentrations in the presence of S9 mix.

Conclusions:

The test substance shows no mutagenic potential to S. typhimurium and E. coli strains.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: Guideline of the Ministry of Health, Labour and Welfare, Japan

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Lot No.: FBX01
Purity: 99.5 %

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 were obtained from Dr. B.N. Ames (University of California), and Escherichia coli strains WP2/pKM101 and WP2uvrA/pKM101 were from Dr. M. Ishizawa (Kyushu University)
- Culturation: inoculated to nutrient broth and incubated for 10 h1's at 37°C in a shaking water bath befo1'e starting the test.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: [yes/no]
- Periodically checked for Mycoplasma contamination: [yes/no]
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]

Species / strain / cell type:

other: S. typhimurium TA 102, TA 104 and E. coli WP2uvr

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA: 0.0763, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate
Experiment 2: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA: 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 µg/plate
Experiment 3: S. typhimurium TA 102, TA 104 and E. coli WP2uvrA/pKM101: 0.0763, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate
Experiment 4: S. typhimurium TA 102, TA 104 and E. coli WP2uvrA/pKM101: 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 µg/plate

Vehicle / solvent:

Vehicle used: Distilled H2O

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrγlamide (AF2)

Remarks:

TA100, TA98 and WP2urA/pKM101 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

WP2/pKM101 without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA98, TA100, TA102, TA104, TA1535, TA1537, WP2/pKM101 and WP2uvrA/pKM101 with S9 mix

Details on test system and experimental conditions:

- Preincubation Method:
The test compound dissolved in 0.05 or 0.1 mL of solvent was supplemented with 0.5 mL of S9 mix (metabolic activation method) or 0.1 M phosphate buffer pH 7.4 (direct method) and 0.1 mL of tester strains which had been cultured in nutrient broth. The mixture was incubated for 20 min at 37°C, and then mixed with 2 mL of molten top algar containing 0.05μmol/ mL of L-histidine and biotin for the Salmonella test (for the E. coli test 0.05μmol / mL of- L-tryptophan was used instead of L-histidine and biotin). Then the top agar mixture was immediately poured onto a 30 mL of Vogel-Bonner minimal glucose agar medium plate. A plate was kept on the flat table and top agar mixture was solidified. All plates were incubated for 48 hrs at 37 °C and the numbers of revertant colonies were scored. Tests on all test chemicals were done in duplicate plates, and those on positive control and solvent control were done in duplicate and quadruplicate plates, respectively.
- Determination of Toxicity (Growth Inhibition):
The growth of the background lawn of tester strains on each plate was examined under a stereo microscope.

Evaluation criteria:

The chemicals were considered to be mutagenic when a dose-related increase in revertant colony count was observed and the number of revertant colonies per plate with the test substance was more than twice that of the negative control and when a reproducibility of test result was observed.

Key result

Species / strain:

S. typhimurium, other: TA104

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: S. typhimurium TA98, TA100, TA102, TA1535, TA1537, E. coli WP2uvrA and WP2uvrA/pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Five experiments were conducted, the test item showed negative results to S. typhimurium TA98, TA100, TA102, TA1535, TA1537, E. coli WP2uvrA and WP2uvrA/pKM101 strains with and without metabolic activation, cytotoxicity observed at 2500 and 5000 μg/plate levels.
For TA 104, three experiments (3, 4, 5) were conducted with and without S9 mix. It showed positive result in experiment 3 with S9 only at 1250 μg/plate, and in experiment 5 with/without S9 at 800 and 1000 μg/plate levels, but negative at levels above 1000 μg/plate. Cytotoxicity observed at 5000 μg/plate level. The increase of revertant colony count is not dose-related.

Conclusions:

The test item showed positive to S. typhimurium TA104, and negative to other S. typhimurium (TA98, 100, 102, 1535, 1537) and E. coli (WP2uvrA, pKM101) strains.

Executive summary:

Bacterial mutagenicity test of test item was performed according to the guideline of the Ministry of Health, Labour and Welfare, Japan.

S. typhimurium TA98, TA100, TA102, TA104, TA1535, TA1537, E. coli WP2uvrA and WP2uvrA/pKM101 were tested with or without S9 mix.

Five experiments were conducted, the test item showed negative results to S. typhimurium TA98, TA100, TA102, TA1535, TA1537, E. coli WP2uvrA and WP2uvrA/pKM101 strains, cytotoxicity observed at 2500 and 5000 μg/plate levels.

For TA 104, three experiments (3, 4, 5) were conducted with and without S9 mix. It showed positive result in experiment 3 with S9 only at 1250 μg/plate, and in experiment 5 with/without S9 at 800 and 1000 μg/plate levels, but negative at levels above 1000 μg/plate. Cytotoxicity observed at 5000 μg/plate level. The increase of revertant colony count is not dose-related.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: "Standard for Chromosomal Aberration Test in Cultured Mammalian Cells" of the Notification No.652 of the Labour Standards Bureau, Ministry of Labour, Japan

Version / remarks:

September 29, 1997

Deviations:

no

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Lot No.: FBX01
Purity: 99.5%

Species / strain / cell type:

other: Chinese hamster lung cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: supplied by Dr. M. Ishidate, Jr. in 1985
- Modal number of chromosomes: 25
- Doubling time: approximately 15 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle's MEM supplemented with 10% heat-inactivated (56°C, 30 min) calf serum

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

24 and 48 h treatment: 0.015, 0.03, 0.06, 0.09, 0.12, 0.15 mg/mL
6h treatment: 0.05, 0.1, 0.2, 0.3, 0.4 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was soluble in DMSO and insoluble in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with and without S9

Details on test system and experimental conditions:

- Short-period treatment with and without Metabolic Activation (6 hours treatment):
Twenty thousands cells were seeded in culture vessel (with 5 mL of culture medium) and cultured for three days, then culture medium was changed. Cells were simultaneously treated with and without S9 mix and the test substance solution for 6 hours. Then, the test substance mixture was changed to fresh culture medium. After 18 hours further culture, rates of cell growth inhibition were determined.
- Continuous Treatment without Metabolic Activation (24 and 48 hours treatment):
Twenty thousands cells were seeded in a culture vessel (with 5 mL of culture medium), and cultured for three days. Then culture medium was changed and the test substance solution was added to a culture medium. After 24 or 48 hours treatment, rates of cell growth inhibition were determined.

- Determination of Cell Growth Inhibition Rate
The medium was discarded and the cells were washed with physiological saline. Ethanol was added to fix the cells which were then stained with 0.1% crystal violet. After washing and drying, each dish was placed under a cell densitometer to measure the color absorption value (550nm). The cell growth index was calculated with the negative control being 100% and Petri dish without cells being 0%.
- Slide preparation:
Two hours before the end of culture, the cells were treated with 0.2μg/mL Colcemid. After finishing culture, they were dissociated with trypsin (0.1% trypsin + 0.036% EDTA) and centrifuged (1000 rpm, 5 min) to collect cells. After removal of the supernatant, the cells were incubated in a 75 mM hypotonic KCl solution for 20 minutes at 37 °C. The cells were then fixed three times with acetic acid-ethanol (1:3) and spread onto clean glass slides. Each slide was stained with Giemsa solution (2.5% at pH 6.8) for 12 minutes after air-drying.
- Observation:
The number of cells with structural aberrations in 100 well-spread metaphase cells were counted per each culture vessel. The incidence of polyploid cells were recorded simultaneously.

Evaluation criteria:

Negative: frequencies of structural aberrations and of polyploidy less than 5%
Equivocal: frequencies of structural aberrations and of polyploidy from 5% to less than 10%
Positive: frequencies of structural aberrations and of polyploidy 10% or more

Key result

Species / strain:

other: Chinese hamster lung cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative result got for test item in chromosomal aberration test in CHL.

Executive summary:

In vitro mammalian chromosome aberration test for test item was performed using lung cells of a newborn Chinese hamster according to "Standard for Chromosomal Aberration Test in Cultured Mammalian Cells" of the Notification No.652 of the Labour Standards Bureau, Ministry of Labour, Japan. The dose level were 0.015, 0.03, 0.06, 0.09, 0.12, 0.15 mg/mL for 24 and 48 h treatment without S9 mix activation, 0.05, 0.1, 0.2, 0.3, 0.4 mg/mL for 6h treatment with and without S9 mix activation. The recovery time forshort-period treatment (6h) was 18 h.

Negative result was given under the test condition.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Two Ames studies are available.

One showed negative to S. typhimurium (TA98, 100, 1535, 1537) and E. coli (WP2) strains both with and without metabolic activation.

In another study, five experiments were conducted, the test item showed negative results to S. typhimurium TA98, TA100, TA102, TA1535, TA1537, E. coli WP2uvrA and WP2uvrA/pKM101 strains with and without metabolic activation, cytotoxicity observed at 2500 and 5000 μg/plate levels. For TA 104, three experiments (3, 4, 5) were conducted with and without S9 mix. It showed positive result in experiment 3 with S9 only at 1250 μg/plate, and in experiment 5 with/without S9 at 800 and 1000 μg/plate levels but negative at levels above 1000 μg/plate. Cytotoxicity observed at 5000 μg/plate level. The increase of revertant colony count is not dose-related.

One in vitro chromosome aberration study showed negative result to CHL cells.

All of these in vitro studies were non-GLP and according to national standard methods which similar to OECD Guidelines.

The results of two Ames studies are consistent as both of them showed negative result to same strains, except for the positive result on strain TA104 in the second study. The positive result on strain TA104 were observed in relatively medium levels but not at high levels, and the increase of revertant colony count is not dose-related, also considering the evidence of cytotoxicity at high doses, we cannot conclude a positive result simply.

Justification for classification or non-classification

In accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, and Guidance on application of CLP criteria section 3.5.2.4, classification Category 2 is based on positive evidence from in vivo/vitro mammalian experiments, in vitro results can only lead to a Category 2 mutagen classification in a case where there is support by chemical structure activity relationship to known germ cell mutagens.

This substance showed positive in Ames study but negative in in vitro mammalian chromosome aberration study, and there is no evidence of analogue chemical which known as mutagen, so this substance shall not be classified for the mutagenicity endpoint.