Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A lot of in vitro studies are available on nitrosodiphenylamine. The most relevant experiment data are presented in this IUCLID.

The response has been negative for the majority of gene mutation studies (Ames and MLA/TK showed negative results). However, nitrosodiphenylamine showed positive response in the standard chromosomal aberration test in the presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Test chemical was tested for mutagenicity in salmonella typhimurium, using a preincubation protocol. All tests were performed in the absence of exogenoux metabolic activation, and in the presenceof liver S-9 from Aroclor-induced male Sprague Dawley rats and Syrian hamsters.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 97
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% or 30% of liver rat or hamster S9
Test concentrations with justification for top dose:
0, 1000, 3300, 10000, 33000, 100000, 200000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine (-S9, TA98)? 2-aminoanthracene (+S9, all strains)
Details on test system and experimental conditions:
Prepration of liver S9 fraction :
The S9 fractions of Aroclor 1254-induced, male SD rat ad male syrian hamster livers were prepared immediately prior to se and contained either 10% or 30% S9; occasionally, other levels were used. All chemicals were tested in the absence of metabolic activation, and with rat and hamster S9 fractions.

Test protocol :
The preincubation assay was performed as follow : the test chemical (0.05 ml), Salmonella culture (0.10 ml), and S9 mix or buffer (0.5 ml) were incubated at 37°C, without shaking for 20 min. The top agar was added and the contents of the tubes were mixed and poured onto the surfce of petri dishes containing medium. The histidine-independent (his +à colonies arising on these plates were counted following two days incubation at 37°C. Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the background agar. At the discretion of the investigators, plates with low numbers of colonies were counted by hand.
All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for mutagnicity assay. The toxicity assay was performed using TA100. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.
Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in the preliminary test. Chemicals that were not toxic were tested to a miximum dose of 10 mg/plate. Chemicals that were poorly soluble were tested up to doses defined by their solubilities. At least 5 doses of each chemical were tested in triplicate. Experiments were repeated at least one week following the initial trial. A maximum of 0.05 ml solvent was added to each plate.

Evaluation criteria:
Evaluationswere made at both the individual trial and overall chemical levels. Individual triales were judged as mutegnic or not depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response.
Statistics:
no
Species / strain:
S. typhimurium, other: all strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
see tables below

Tables of results (tables 207 : nitrosodiphenylamine)

Dose

(µg/ plate)

TA100

Without S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

82

6.2

95

9.7

86

24.7

209

1.3

95

2.3

1000

84

7.4

 

 

 

 

 

 

 

 

3300

90

9.0

91

3.5

97

6.0

200

8.1

91

6.1

10 000

84

6.2

114

5.0

96

10.2

203

17.3

89

2.0

33 000

86

4.7

101

7.5

92

5.5

206

1.7

96

10.9

100 000

61s

3.6

107

5.7

111

5.7

177

14.4

102

5.9

200 000

 

 

122s

5.0

76s

3.2

158s

2.1

90s

3.6

POS

429

7.6

260

14.2

363

16.5

424

17.9

323

16.4

HLI : hamster liver ; RLI : rat liver

Dose

(µg/ plate)

TA1535

Without S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

22

0.9

16

1.8

7

3.2

21

1.7

11

2.4

1000

23

1.0

 

 

 

 

 

 

 

 

3300

27

6.1

13

1.9

10

1.8

17

1.8

10

0.6

10 000

30

4.4

12

2.3

5

1.3

18

4.3

10

1.5

33 000

37

5.6

18

2.8

8

1.8

16

0.9

9

0.6

100 000

7s

2.0

12

1.2

12

1.7

18

2.0

14

1.8

200 000

 

 

15s

3.7

9s

3.2

17s

1.2

11s

0.3

POS

263

11.2

86

10.6

219

13.4

222

11.3

163

6.4

HLI : hamster liver ; RLI : rat liver

Dose

(µg/ plate)

TA1537

Without S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

8

2.2

9

2.0

6

0.7

16

3.2

12

1.5

1000

4

1.2

 

 

 

 

 

 

 

 

3300

5

2.3

9

1.8

8

2.3

12

2.3

12

1.3

10 000

7

0.7

11

1.5

7

0.6

14

1.7

9

1.9

33 000

7

0.9

10

0.9

8

0.0

14

0.6

8

1.5

100 000

4s

1.2

7

1.5

11

1.0

14

2.2

9

1.5

200 000

 

 

8s

2.4

10s

0.6

10s

0.9

9

2.8

POS

126

12.4

121

1.8

111

15.5

161

7.8

55

2.7

HLI : hamster liver ; RLI : rat liver

Dose

(µg/ plate)

TA97

Without S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

81

4.1

210

8.5

128

3.7

214

9.4

170

13.5

1000

76

9.7

 

 

 

 

 

 

 

 

3300

79

1.3

189

7.9

119

4.3

233

1.5

123

2.8

10 000

67

3.1

183

12.0

129

9.2

235

17.8

126

3.5

33 000

76

2.3

201

22.6

126

10.7

245

10.3

130

10.8

100 000

8s

1.5

220

4.8

132

3.9

265

12.5

124

4.4

200 000

 

 

141s

3.5

140

7.0

178s

7.8

130

5.5

POS

235

19.6

543

15.5

696

22.7

873

77.4

398

7.8

HLI : hamster liver ; RLI : rat liver

 

 

Dose

(µg/ plate)

TA98

Without S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

13

1.8

22

1.7

26

5.0

27

4.5

19

1.2

1000

11

1.8

 

 

 

 

 

 

 

 

3300

13

0.3

21

1.7

24

1.7

29

1.2

21

2.1

10 000

10

4.8

25

0.6

18

3.0

24

0.6

20

3.1

33 000

12

2.2

24

4.0

19

2.2

20

2.0

21

1.7

100 000

8s

3.6

25

2.8

27

1.5

21

1.0

20s

2.9

200 000

 

 

19s

1.0

25s

2.2

17s

1.3

22s

2.6

POS

157

12.3

193

6.7

164

24.1

158

8.1

152

16.4

HLI : hamster liver ; RLI : rat liver

 

Conclusions:
Based on this assay, nitrosodiphenylamine showed negative results in the Ames test.
Executive summary:

Test chemical was tested for mutagenicity in Salmonella typhimurium, using a preincubation protocol. All tests were performed in the absence of exogenoux metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague Dawley rats and Syrian hamsters. Based on this assay, nitrosodiphenylamine showed negative results in the Ames test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
no
Type of assay:
other: in vitro chromosomal aberration study in mammalian cells
Target gene:
n/a
Species / strain / cell type:
other: Chinese hamster lung cell line
Details on mammalian cell type (if applicable):
Cell line was established form the lung of a newborn chinese hamster by Dr Utakoji et al. (1970).
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Colcemid
Metabolic activation:
with and without
Metabolic activation system:
liver rat
Test concentrations with justification for top dose:
Without S9 : 0.01, 0.02, 0.04, 0.08, 0.12 mg/ml
With S9: 0.025, 0.05, 0.1, 0.15, 0.2 mg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
The cell was cultured with Eagle's MEM supplemented with 10% heat-inactivated (56°C, 30 min) calf serum in the CO2 incubator. The modal chromosome number was 25 and the doubling time time approximately 15 hours. The cells were cultured in a plastic-flask as monolayer, and sub-cultured at 2-4 days intervals. The cells were stored as freezed state in liquid nitrogen.
The test substance was added to culture medium as homogeneous solution. The volumes of medium and test substance solution used in the test system were 0.025 ml and 5.0 mg respectively.

A post-mitochondrial supernatant fraction of liver homogenates (S9, SD rat, inducer : sodium phenobarbital and 5.6-benzoflavone) was obtained commercially from Kikkoman corporation (Japan). S9 mix consists of 3 ml of S9 fraction, 2 ml of 20mM HEPES buffer (pH 7.2), 1 ml of 50 mM MgCl2, 1 ml of 330 mM KCl, 1 ml of 50 mM G-6-P, 1 ml of 40 mM NADP, and 1 ml of distilled water. The S9 mix was prepared immediately prior to use. The S9 was stored at -80°C and used within 6 months after preparation.
The final concentration of the S9 fraction in the final treatment medium was 5%.

CELL GROWTH INHIBITION STUDY
The cell growth inhibition test which is used for checking cytotoxicity was carried out in order to determine the treatment levels of the test substance in the chromosomal aberration test.
Without S9: 2 x 10^4 cells were seeded in a 60 mm Petri dish (with 5 ml of culture medium) and cultured for three days, then the test substance solution was added to a dish of culture medium. After 24 or 48 hours treatment, rates of cell growth inhibition were determined.
With S9: The S9 mix was added to the culture medium for 6 hours treatment. 2 x 10^4 cells were seeded in a 60 mm Petri dish (with 5 ml of culture medium) and cultured for three days. Cells were simultaneously treated with 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.

Determination of cell growth inhibition rate : In the case of the Petri dish culture, 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 (555 nm). The cell growth index was calculated with the negative control being 100%, and Petri dish without cells being 0%.

CHROMOSOMAL ABERRATION TEST
Chromosomal aberration test was carried out at several (five) concentrations of test substance selected from the result of cell growth inhibition study. Two culture vessels wer used for each concentration of test substance.
The cells were treated with 0.2 µg/ml of colcemid for 2 hours, and after treatment with trypsin they were incubated in 75 mM hypotonic KCl for 20 minutes at 37°C. The cells were fixed with acetic acid-ethanol (1:3) and spread onto clean glass slides. After air-drying, each slide was stained with Giemsa solution (2.5% at pH 6.8) for 12 minutes.
The number of cells with chromosomal aberrations from 100 metaphases were counted per each culture bottle. The types of aberrations were classified into 6 groups : chromatid and chromosomes gaps (g), chromatid breaks (ctb), chromatid exchange (cte), chromosome breaks (csb), chromosome exchange (sce), and others (fragmentation..). The incidence of polypoil cells in the 100 metaphases was recorded.


Evaluation criteria:
For the evaluation of the frequencies of structural aberrations and of polyploidy, the following criteria which are usually used for chromosomal aberrations testing with CHL are employed :
Negative : less than 5%
Equivocal : from 5% to less than 10%
Positive : 10% or more
The total frequencies of structural aberrations include the frequencies of aberrant cells with have only gaps.
Statistics:
no
Species / strain:
mammalian cell line, other: CHL
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mammalian cell line, other: CHL
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
see the tables of results

Table 1 of results: polyploidy (without S9)

Treatment

No. of metaphase

Polyploidy (%)

Judgement

Substance

Time (h)

Concentration

DMSO

24

 

200

1.0

Negative

48

 

200

0.0

Negative

Test chemical

24

0.01

200

0.0

Negative

0.02

200

1.0

Negative

0.04

200

0.0

Negative

0.08

200

0.0

Negative

0.12

200

0.5

Negative

48

0.01

200

0.5

Negative

0.02

200

0.0

Negative

0.04

200

1.0

Negative

0.08

200

2.5

Negative

0.12

200

0.0

Negative

Positive control

24

0.00004

200

2.0

Negative

48

0.00004

200

1.0

Negative

 

Table 2 of results: structural chromosome aberration (without S9)

Treatment

No. of metaphase

Cell with structural chromosome aberrations (%)

Judgement

gap

Chromatid

Chromosome

Total

Substance

Time (h)

Concen-tration

CTB

CTE

CSB

CSE

-G

+G

DMSO

24

 

200

0

0.5

0

0

0

0.5

0.5

Negative

48

 

200

0

0

0

0

0

0

0

Negative

Test chemical

24

0.01

200

0

0

1.0

0

0

1.0

1.0

Negative

0.02

200

0

0.5

0

0

0

0.5

0.5

Negative

0.04

200

0

1.5

1.5

0

0

3.0

3.0

Negative

0.08

200

0.5

0.5

0.5

0

0

1.0

1.0

Negative

0.12

200

0.5

2.5

2.0

0

0

4.0

4.0

Negative

48

0.01

200

0

0

1.0

0

0

1.0

0

Negative

0.02

200

0

0

0

0

0

0

2.0

Negative

0.04

200

0

0

0

0

0

0

5.4

Negative

0.08

200

0

0.5

0.5

0

0

1.0

1.0

Negative

0.12

200

0

0.5

2.0

0

0

2.5

2.5

Negative

Positive control

24

0.00004

200

1.5

8.0

48.0

0

0.5

51.0

51.0

Positive

48

0.00004

200

2.5

14.5

71.0

0

0

73.0

73.0

Positive

 

 

 

 

 

 

 

 

 

 

Table 3 of results: polyploidy (with S9)

Treatment

No. of metaphase

Polyploidy (%)

Judgement

Substance

Time (h)

Concentration

DMSO

24

 

200

2.0

Negative

48

 

200

0.5

Negative

Test chemical

24

0.025

200

1.0

Negative

0.05

200

1.0

Negative

0.1

200

0.5

Negative

0.15

No observation for metaphase

0.2

No observation for metaphase

48

0.025

200

0.5

Negative

0.05

200

0.0

Negative

0.1

200

1.5

Negative

0.15

200

0.5

Negative

0.2

No observation for metaphase

Positive control

24

0.01

200

1.0

Negative

48

0.01

200

0.0

Negative

 

Table 4 of results: structural chromosome aberration (with S9)

Treatment

No. of metaphase

Cell with structural chromosome aberrations (%)

Judgement

gap

Chromatid

Chromosome

Total

Substance

Time (h)

Concen-tration

CTB

CTE

CSB

CSE

-G

+G

DMSO

24

 

200

0

0

0.5

0

0

0.5

0.5

Negative

48

 

200

0

0

1.5

0

0

1.5

1.5

Negative

Test chemical

24

0.025

200

0

0

1.0

0

0

1.0

1.0

Negative

0.05

200

0

0.5

2.5

0

0

3.0

3.0

Negative

0.1

200

0

0.5

1.5

0

0

1.5

1.5

Negative

0.15

No observation for metaphase

0.2

No observation for metaphase

48

0.025

200

0

0

1.0

0

0

1.0

1.0

Negative

0.05

200

0.5

4.0

13.5

0

0

15.0

15.5

Positive

0.1

200

1.0

7.0

36.5

0

0

40.5

40.5

Positive

0.15

200

1.5

7.0

21.0

0

0

25.5

25.5

Positive

0.2

No observation for metaphase

Positive control

24

0.01

200

0.5

1.0

0.5

0

0

2.0

2.0

Negative

48

0.01

200

0

2.5

25.0

0

0

27.0

27.0

Positive

 

Conclusions:
Based on these experimental data, the test substance induced chromosomal aberrations in the presence of metabolic activation only.
Executive summary:

Chromosomal aberration test in CHL cell was performed with nitrosodiphenylamine. The test was carried out 5 concentrations of test substance selected from the results of cell growth inhibition study. Two cultures were used for each concentration of test substance. The test was performed without metabolic activation (after 24 and 48 hours treatment), and with liver-rat metabolic activation (after 6 hours of treatment). 100 metaphases were counted per culture.

Based on these experimental data, Nitrosodiphenylamine induced chromosomal aberrations in the presence of metabolic activation only.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The L5178YTK+/- is a short-term test to identify chemicals induced forward mutation in cultured mammalian cells.
GLP compliance:
no
Type of assay:
other: in vitro gene mutation test in mammalian cells.
Specific details on test material used for the study:
the chemical was supplied by ICN-K and K laboratories (Clevelan, OH).
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Heterozygous L5178Y TK+/- 3.7.2C cells were obtained from Dr. Dnarld Clive (Burroughs Welcome Co.).
- Periodically checked for Mycoplasma contamination: yes>> free of contamination
Metabolic activation:
with and without
Metabolic activation system:
rat liver (Fisher 344, induced by Arochlor 1254)
Test concentrations with justification for top dose:
Without S9: 0, 75, 50, 25? 10, 1 µg/ml
With S9 : 0, 10, 8, 6, 4, 2, 1, 0.5 µg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Details on test system and experimental conditions:
Chemical was prepared and diluted in DMSO, and 0.1ml of each dilution was added to a 10-ml suspension containing 6 X 10¨6 cells from a culture recently cleansed of TK+/- cells.
For the activated portion of the assay, the 10 ml suspension included 4 ml of S9 and cofactor mixture. All cutures prepared for testing contained 50% conditioned media and 50% fresh medium without serum. The serum concentration was further reduced to 3% during the period of chemical exposure by the addition of serum or S9. Following a 4-h exposure to the test chemical, the cells were washed twice, fresh medium was added, and the cultures were incubated for the 2-day expression period.
To determine TK-/- mutants, approxmately 5 x 10^5 viable cells for each treatment were cloned in triplicate using selective edium which contained 3 µg/ml trifluorothymidine (TFT).
To determine the cloning efficiency, 100 cells for each treatment were cloned in triplicate in non-selective medium.
Cloning media consisted of RPMI 1640 with 20% horse serum and 0.40% Noble agar. Cell inocular were added directly into 100-mm tissue culture plates, followed by the addition of about 30 mlof either selective and non-selective cloning medium. The plates were swirled gently yo ensure even dispersal of the inocula, allowed to gel at room temperature for approx. 15 min and then incubated at 37°C for 11 days prior to colony counting.
Evaluation criteria:
A positive response was demonstrated by a dose-related increase in mutation frequency in which the mutation frequency of treated cultures exceeded that of solvent-control cultures by at least 2-fold for a minimum of 2 successive concentrations where values for total survival exceeded 10%.
Statistics:
no
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Table of results :

Metabolic activation

Dose (µg/ml)

Total survival

(a)

Mutation frequency (b)

Mutation index (c )

WITHOUT

0

100

1.9

-

75

14

2.6

1.4

50

25

3.3

1.7

25

54

2.4

1.3

10

84

1.5

0.8

1

74

2.1

1.1

WITH

0

100

3.6

-

10

2

7.0

1.9

8

4

5.2

1.4

6

5

4.6

1.3

4

13

6.1

1.7

2

25

4.0

1.1

1

36

3.3

0.9

0.5

38

4.7

1.3

(a) = suspension growth x cloning efficiency

(b) = Mutants / 10^5 surviving cells

(c ) = Mutation frequency of treated culture / mutation frequency of control culture

Executive summary:

The L5178Y TK+/- is a short-term test to identify chemicals induced forward mutation in cultured mammalian cells.

The assay was performed with and without rat metabolic activation.

Nitrosodiphenylamine showed negative results in this assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo animal studies involving mice and rats consistently show negative results for DNA damage (Brambilla 1987), micronuclei (Salomone 1981, Tsuchimoto 1981) and DNA synthesis inhibition (Friedman 1976).

In conclusion, Nitrosodiphenylamine does not appear to be genotoxic in intact animal system.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A new viscometric technique, capable of detecting DNA strand breaks and alkali-labile sites by monitoring time-dependent changes of DNA-reduced viscosity, has been used to analyse dose-response curves for the induction of DNA damage in liver of rats treated with single p.o. doses.
The contemporary measurement of liver DNA fragmentation by the alkaline elution technique revealed hat in the experimental conditions higher doses are needed to produce statistically significant increase of DNA elution rate. This suggests that the viscometric method is capable of detecting smaller levels of N-nitroso compound induced DNA fragmentation.
GLP compliance:
no
Type of assay:
other: DNA fragmentation
Specific details on test material used for the study:
Abbreviation in the publication : NDPHA
molecular weight : 198.2 g/mol
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
- Weight at study initiation: 160-180g
- Assigned to test groups randomly: yes
- Fasting period before study: 12h

Route of administration:
oral: gavage
Vehicle:
Nitrosodiphenylamine was suspended in distilled water with 1% carboxymethyl cellulose
Details on exposure:
A single dose was administered by gavage in 0.01 ml of vehicle/g of body weight.
Duration of treatment / exposure:
only one treatment
Frequency of treatment:
only one treatment
Post exposure period:
Rats were sacrified 3h after treatment.
Dose / conc.:
540 mg/kg bw/day
Remarks:
corresponding of one-third of LD50
No. of animals per sex per dose:
Control groups : 20 rats
Treated groups : 3 rats
Control animals:
yes, concurrent vehicle
Positive control(s):
no
Tissues and cell types examined:
Liver cells
Details of tissue and slide preparation:
Liver cell nuclei of treated and control rats were obtained by liver perfusion, and analyzed viscometrically in alkaline conditions (pH 12.5) at high ionic strength (I = 1.08). The cells harvested with the procedure used were predominantly of the lobular prenchyma. The number of nuclei lysed in the circular channel of the viscometer ranged from 6 to 7 x 10^7.
Results are expressed as reduced viscosity that is the viscosity of DNA solution for conentration units of DNA, determined at the end of each experiment.
To obtain quantitative parameters, viscosity measurements were performed at 20 and 40-min intervals.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not examined
Additional information on results:
Fragmentation of rat liver DNA :
DNA damage was absent in the liver of rats given 540 mg/kg of nitrodiphenylamine.
% DNA eluted from the filter : 19.2 +/-6.3
DNA elutation rate over controls : 0.0020

Effet of nitrosodiphenylamine on viscometric behavior of rat liver DNA :
The time required for DNA reduced viscosity to reach 95% = T95 = 1411 min +/-88 min
Reduced viscosity at 95% = 3.05 +/-0.11 (x10-2 dl/g)
Slope = 0.22 +/- 0.03 x 10-2
Conclusions:
DNA damage was undetectable in liver of rats treated with 540 mg/kg of nitrosodiphenylamine, that exhibited a plateau effect.
Executive summary:

A new viscometric technique, capable of detecting DNA strand breaks and alkali-labile sites by monitoring time-dependent changes of DNA-reduced viscosity, has been used to analyse dose-response curves for the induction of DNA damage in liver of rats treated with single p.o. doses.

The contemporary measurement of liver DNA fragmentation by the alkaline elution technique revealed hat in the experimental conditions higher doses are needed to produce statistically significant increase of DNA elution rate. This suggests that the viscometric method is capable of detecting smaller levels of N-nitroso compound induced DNA fragmentation.

With both techniques, DNA damage was undetectable in liver of rats treated with 540 mg/kg of nitrosodiphenylamine, that exhibited a plateau effect.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
Modified bone marrow micronucleus assay involved sampling one dose level at multiple times rather than testing multiple doses at a single time.
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
not specified
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: From Biobreeders Laboratory, Ottawa, Ontario
- Age at study initiation: no data
- Weight at study initiation: no data
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: 5 or 15 by cage
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum):no data
- Acclimation period: 1 / 2 weeks


ENVIRONMENTAL CONDITIONS :
-light : 12h by day from 6.00
Route of administration:
intraperitoneal
Vehicle:
DMSO
Details on exposure:
In the first phase, mice were injected intraperitoneally with the agent at 0 and 24 hr, and samples were taken at 48, 72, and 96 hr. Each treatment consisted of a dose equal to 80% of the LD50/7 (the dose required to kill 50% of the animals within 7 days). If there was a significant increase in the frequency of micronuclei at any sample time, then the treatment was repeated and animals sampled at the appropriate time or a graded series of doses were tested at the appropriate sample time. In either case, the agent was classified as clastogenic if there was a confirmation of the initial positive response, and no further testing was performed. If no increase in the micronucleus frequency was detected in phase 1 or in the confirmation test, then a single treatment of either 50% or at both 80% and 40% of the LD50/7 was given and samples were taken at 30, 48, and 72 hr (phase 2). Where the response was negative for both phases, the agent was classified as nonclastogenic. However, when an increase in the frequency of micronuclei was noted in phase 2, a confirmation test was then performed.
Duration of treatment / exposure:
2 days
Frequency of treatment:
1/daily
Post exposure period:
3 days
No. of animals per sex per dose:
5 mice / sample time
Control animals:
not specified
Positive control(s):
yes : cyclophosphamide
Tissues and cell types examined:
Bone marrow cells were examined.
Details of tissue and slide preparation:
no
Evaluation criteria:
Criteria in testing protocol upon which a positive results is not based on statistical difference between negative control (not present in the study).
Each group represents a dose-time point. If the total number of micronuclei within a group for a given number of mice or exceeds that value shown below, then the response is considered positive. Criteria are based upon 500 PCE counted per mouse.
5 mice per group / experiment with only one experiment : threshold of MN = 6 ; 4 mice = 5 MN ; 3 mice = 4 MN
4 mice per group / experiment with two experiments : threshold of MN = 6 ; 3 mice = 5 MN
Statistics:
Analysis was based on the distribution of micronuclei among mice (when 500 PCE were scored for each mouse) that showed no significant deviation from the Poisson distribution. At that time, 111 micronuclei had been seen in 173 control mice, so that the mean frequency per mouse was 0.64-2. We decided to accept 10% false positives in an experiment, so that the probability that a chemical would be a false positive in two successive experiments and would thus be (falsely) classified as a confirmed positive would be 1%. False positives that are confirmed can also arise among those groups in which the first and second experiments do not agree and a third is used for a decision.
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
not valid
Additional information on results:
Diphenylnitrosamine was negative in either phase 1 and phase 2, so was considered to be not clastogenic.

Individual test results for diphenylnitrosamine :

 

Dose

%LD50

No of treatment

Sampling time

36h

48h

72h

96h

Phase 1

80

2

 

0.0.0.0.0

0.0.1.1

0.0.0

Phase 2

80

1

0.0.2.2

0.0.1

 

 

Phase 2

50

1

0.0.0

0.1.2

 

 

 

Conclusions:
Diphenylnitrosamine was negative in either phase 1 and phase 2, so was considered to be not clastogenic.
Executive summary:

In the first phase, mice were injected intraperitoneally with the agent at 0 and 24 hr, and samples were taken at 48, 72, and 96 hr. Each treatment consisted of a dose equal to 80% of the LD50/7 (the dose required to kill 50% of the animals within 7 days). If there was a significant increase in the frequency of micronuclei at any sample time, then the treatment was repeated and animals sampled at the appropriate time or a graded series of doses were tested at the appropriate sample time. In either case, the agent was classified as clastogenic if there was a confirmation of the initial positive response, and no further testing was performed. If no increase in the micronucleus frequency was detected in phase 1 or in the confirmation test, then a single treatment of either 50% or at both 80% and 40% of the LD50/7 was given and samples were taken at 30, 48, and 72 hr (phase 2). Where the response was negative for both phases, the agent was classified as nonclastogenic. However, when an increase in the frequency of micronuclei was noted in phase 2, a confirmation test was then performed.

Diphenylnitrosamine was negative in either phase 1 and phase 2, so was considered to be not clastogenic.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The ability of test chemical to increase the frequency of micronucleated erythrocytes in CD-1 mice was determined.
GLP compliance:
no
Type of assay:
other: in vivo micronucleus assay
Specific details on test material used for the study:
Name of the compound in the article : Diphenylnitrosamine (substance n°17).
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River, Paris.
Route of administration:
intraperitoneal
Vehicle:
DMSO
Details on exposure:
Test substance was administered i.p. twice, 24 hr apart. The animals were killed 6 hr after the second application.
Duration of treatment / exposure:
n/a : injection
Frequency of treatment:
twice, 24h apart
Post exposure period:
6 hr after the second application
Dose / conc.:
125 mg/kg bw/day
Remarks:
= 1/8 LD50 (1000-1100)
Dose / conc.:
250 mg/kg bw/day
Remarks:
= 1/4 LD50 (1000-1100)
Dose / conc.:
500 mg/kg bw/day
Remarks:
= 1/2 LD50 (1000-1100)
No. of animals per sex per dose:
2 males and 2 femelles per group
Control animals:
yes, concurrent vehicle
Positive control(s):
yes : B(a)P, aminofluorene, cyclophosphamide
Tissues and cell types examined:
Femoral bone marrow cells were obtained and swears were prepared.
Details of tissue and slide preparation:
For staining the slides, phosphate buffer (pH 6.88) was used insteadof distilled water. All the slides were coded.
An analysis of the smears was carried out in the following way. Screening of the slides was performed, using 10 x 16 magnification, for regions where cells were well spread and optimally stained. 1500 polychromatic erythrocytes per animal were analyzed under oil-immersion high power magnification ( 10 x 100). The number of micronucleated polychromatic erythrocytes (MPEs) were counted, but not the number of micronuclei.
Evaluation criteria:
The data were evaluated on the basis of the following criteria :
1) two or more mice per group with MPE frequencies above 0.40%,
2) one or more treated groups with mean MPE frequencies above 0.30%, and,
3) statistical significance in one or more treated groups.
A test substance was judged positive when all three of these criteria were met, negative when they were not, and questionable when one or two were met.
Statistics:
yes
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
see table of results

 

 

Table of results: percentage of micronucleated polychromatic erythrocytes :

Control

Low dose group

125 mg/kg

Mid-dose group

250 mg/kg

High dose group

500 mg/kg

0.10

(0.00-0.20)

0.12

(0.00-0.20)

0.15

(0.07-0.40)

0.07

(0.07-0.07)

Conclusions:
The test substance was negative in this in vivo micrunucleus assay, so was considered to be not clastogenic.
Executive summary:

The ability of test chemical to increase the frequency of micronucleated erythrocytes in CD-1 mice was determined. Test substance, disolved in DMSO, was administered i.p. twice, 24 hr apart, to 2 males and 2 femelles per group. Four groups were used in this assay : 0, 125, 250, 500 mg/kg. The animals were killed 6 hr after the second application. Femoral bone marrow cells were obtained and swears were prepared. 1500 polychromatic erythrocytes per animal were analyzed under oil-immersion high power magnification ( 10 x 100). The number of micronucleated polychromatic erythrocytes (MPEs) were counted.

Nitrosodiphenylamine was negative in this in vivo micrunucleus assay, so was considered to be not clastogenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

It is widely accepted that no single short-term test as an adequate predictor of genotoxicity and therefore batteries of test is required.

Several Ames test are available. Based on the weight of evidence, Nitrosodiphenylamine is considered to not mutagenic in S.typhimurium. There is no reproducible effect upon strains of E.coli.

Zeiger (1988): five strains (S.typhimurium TA1535, TA1537, TA97, TA100, TA98), +/- S9 of rat or hamster (10% or 30%): Negative

Crebelli (1984): Two strains (S.typhimurium TA100, TA98), +/-S9 (rat ; 10% of S9): Negative

Richold (1981): Five strains (S.typhimurium TA1535, TA1537, TA1538, TA100, TA98), +/-S9 (rat ; 10% of S9): Negative

Venitt (1981): 4 strains (S.typhimurium TA98 and TA100, E.coli WP2 uvrA(P) and WP2(P), +/- S9 (rat ; 4, 10 and 30%) : Ambiguous results.Negative without S9, Ambiguous with S9 (TA100 : positive with 10% of S9 but negative with 4 and 30 % S9 ; E.coli WP2 : positive with 4% S9 but negative with 10 and 30% S9), Positive with S9 (E.coli WP2 uvrA : 10 and 30% S9)

Dunkel (1979): 6 strains (TA1535, TA1536, TA1537, TA1538, TA100 and TA98), +/-S9 (rat ; % not precised): Negative

A mouse lymphoma TK is negative with and without S9 (Oberly 1984). The L5178Y TK+/- is a short-term test to identify chemicals induced forward mutation in cultured mammalian cells. The assay was performed with and without rat metabolic activation. Nitrosodiphenylamine showed negative results in this assay.

 

Two reliable in vitro chromosomal aberration tests are available on nitrosodiphenylamine:

JETOC (1997): standard test on CHL cells +/- S9: Positive with S9, negative without S9.

Chromosomal aberration test in CHL cells was performed with nitrosodiphenylamine. The test was carried out 5 concentrations of test substance selected from the results of cell growth inhibition study. Two cultures were used for each concentration of test substance. The test was performed without metabolic activation (after 24 and 48 hours treatment), and with liver-rat metabolic activation (after 6 hours of treatment). 100 metaphases were counted per culture.

Based on these experimental data, Nitrosodiphenylamine induced chromosomal aberrations in the presence of metabolic activation only.

Dean (1981): non-standard test on liver rat cells without S9: Negative

 

Some in vitro DNA damage assays were available on nitrosodiphenylamine but the results are not concordant.

Althaus (1982): assay on rat hepatocytes, without S9 : Positive

Probst (1980): assay on rat hepatocytes, without S9 : Negative

Four in vivo studies are available on Nitrosodiphenylamine, and allowed to conclude that Nitrosodiphenylamine is not mutagenic in intact animals.

Tsuchimoto (1981): in vivo micronucleus assay in mice treated by i.p. : Negative.

The ability of test chemical to increase the frequency of micronucleated erythrocytes in CD-1 mice was determined. Test substance, dissolved in DMSO, was administered i.p. twice, 24 hr apart, to 2 males and 2 femelles per group. Four groups were used in this assay : 0, 125, 250, 500 mg/kg. The animals were killed 6 hr after the second application. Femoral bone marrow cells were obtained and swears were prepared. 1500 polychromatic erythrocytes per animal were analyzed under oil-immersion high power magnification. The number of micronucleated polychromatic erythrocytes (MPEs) were counted. Nitrosodiphenylamine was negative in this in vivo micronucleus assay, so was considered to be not clastogenic.

Salomone (1981): in vivo micronucleus assay in mice treated by i.p. : Negative.

In the first phase, mice were injected intraperitoneally with the agent at 0 and 24 hr, and samples were taken at 48, 72, and 96 hr. Each treatment consisted of a dose equal to 80% of the LD50/7 (the dose required to kill 50% of the animals within 7 days). If there was a significant increase in the frequency of micronuclei at any sample time, then the treatment was repeated and animals sampled at the appropriate time or a graded series of doses were tested at the appropriate sample time. In either case, the agent was classified as clastogenic if there was a confirmation of the initial positive response, and no further testing was performed. If no increase in the micronucleus frequency was detected in phase 1 or in the confirmation test, then a single treatment of either 50% or at both 80% and 40% of the LD50/7 was given and samples were taken at 30, 48, and 72 hr (phase 2). Where the response was negative for both phases, the agent was classified as nonclastogenic. However, when an increase in the frequency of micronuclei was noted in phase 2, a confirmation test was then performed. Nitrosodiphenylamine was negative in either phase 1 and phase 2, so was considered to be not clastogenic.

Brambilla (1987): in vivo DNA damage assay in rat treated by gavage : Negative.

A new viscometric technique, capable of detecting DNA strand breaks and alkali-labile sites by monitoring time-dependent changes of DNA-reduced viscosity, has been used to analyse dose-response curves for the induction of DNA damage in liver of rats treated with single p.o. doses. The contemporary measurement of liver DNA fragmentation by the alkaline elution technique revealed hat in the experimental conditions higher doses are needed to produce statistically significant increase of DNA elution rate. This suggests that the viscometric method is capable of detecting smaller levels of N-nitroso compound induced DNA fragmentation. With both techniques, DNA damage was undetectable in liver of rats treated with 540 mg/kg of nitrosodiphenylamine, that exhibited a plateau effect.

Friedman (1976): in vivo DNA synthsis in mice treated by ip : Negative

Groups of mice were injected i.p. with nitrodiphenylamine and killed 3.5h later. 30 minutes prior killing, each mouse was injected with 10 µCi tritied-thymidine. Control mice received 0.05 ml ethanol and only the thymidine injection. Testes were isolated and DNA specific activity determined.

Nitrosodiphenylamine did not induce structural change in DNA in mice treated with a single dose of 500 mg/kg bw : Nitrosodiphenylamine didn't alter tritied-thymidine uptake into testicular DNA.

Justification for classification or non-classification

A lot of in vitro studies are available on nitrosodiphenylamine. The response has been negative for the majority of gene mutation studies (majority of Ames negative, and negative MLA/TK). However, nitrosodiphenylamine showed positive response in the standard chromosomal aberration test in the presence of metabolic activation.

Most of the positive responses occurred in cased where exogenous metabolic activation was involved. This suggests that if nitrosodiphenylamine has genotoxic potential, the potential may arise from its metabolites.

However, nitrosodiphenylamine does not appear to be genotoxic in intact animal system.

In conclusion, nitrosodiphenylamine is considered to be not mutagenic ; no classification is required according to the Regulation EC n°1272/2008.