N,N'-diphenyl-p-phenylenediamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Aug 2016 to 25 Aug 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-diphenyl-p-phenylenediamine

EC Number:

200-806-4

EC Name:

N,N'-diphenyl-p-phenylenediamine

Cas Number:

74-31-7

Molecular formula:

C18H16N2

IUPAC Name:

N,N'-diphenyl-p-phenylenediamine

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: QB-DBF-001
- Date of production/supply: Jul 2016
- Expiration date of the lot/batch: July 2018
- Purity test date: 35%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Physical state, appearance: solid, white to dark grey
- Solubility and stability of the test substance in the vehicle: To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The stability of a comparable batch (20150925) in the vehicle DMSO has been verified analytically.

Method

Target gene:

S. typhimurium: his
E. coli: trp

Species / strain

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 from phenobarbital (i.p.) and β-naphthoflavone (oral) induced rats livers

Test concentrations with justification for top dose:

JUSTIFICATION
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. In this study, due to the purity of the test substance 14.3 mg/plate was used as top dose.

TEST CONCENTRATIONS
- Without S9 mix: 71.5; 357.5; 715; 3575; 7150; 14300 μg/plate.
- With S9 mix: 71.5; 357.5; 715; 3575; 7150; 14300 μg/plate.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

yes

Remarks:

Sterility control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION
In agar (plate incorporation)

DURATION
- Exposure duration: 47 – 72 hours at 37°C in the dark

NUMBER OF REPLICATIONS
3 plates/dose

DETERMINATION OF CYTOTOXICITY
The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)

Evaluation criteria:

ACCEPTANCE CRITERIA
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10^9 cells per mL were used.

ASSESSMENT CRITERIA
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Decreased revertant numbers:TA 1535, TA 1537, and TA 98 3575 – 14300 μg/plate; TA 100 7150 - 14300 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Decreased revertant numbers at 3575 – 14300 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

See 'Additional information on results'

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Decreased revertant numbers for TA 1535 at 7150 – 14300 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced background growth at 14300 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 715 μg/plate onward with and without S9 mix.

ADDITIONAL INFORMATION ON MUTAGENICITY
- TA 1535: Increase in the number of his+ revertants at a concentration of 71.5 μg/plate (factor 3.1).
- TA 1537: Increase in the number of his+ revertants at concentrations of 7150 and 14300 μg/plate (factors 7.3 and 9.8, respectively).
- TA 98: Slight increase in the number of his+ revertants at a concentration of 7150 μg/plate (factor 1.8).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see Table 1 in section "Any other information on results incl. tables"
- Negative (solvent/vehicle) historical control data: see Table 2 in section "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1. Positive historical control data

Strain	S9 mix	Positive control	Min	Max	Mean	SD
TA 1535	Without	MNNG	1174	6612	4063	1308.8
	With	2-AA	108	367	217	50.0
TA 100	Without	MNNG	1125	5557	3331	1021.5
	With	2-AA	361	2819	1792	448.9
TA 1537	Without	AAC	235	1858	975	339.9
	With	2-AA	56	241	130	33.6
TA 98	Without	NOPD	271	1119	486	183.3
	With	2-AA	431	2427	1427	361.9
E. coli	Without	4-NQO	133	1552	884	442.4
	With	2-AA	54	169	98	26.6

Abbreviations: MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; 2-AA, 2-aminoanthracene; AAC, 9-aminoacridine; NOPD, 4-nitro-o-phenylenediamine; 4-NQO, 4-nitroquinoline-N-oxide

Table 2. Negative historical control data

Strain	S9 mix	Min	Max	Mean	SD
TA 1535	Without	6	17	10	2.2
	With	6	18	10	2.1
TA 100	Without	69	140	95	9.9
	With	76	150	100	12.3
TA 1537	Without	5	12	7	1.5
	With	5	16	8	2.2
TA 98	Without	12	30	18	3.1
	With	13	38	24	4.7
E. coli	Without	12	33	22	4.0
	With	13	35	23	4.0