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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames tests with negative response are available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP conditions
Qualifier:
according to
Guideline:
other: NTP protocols
Deviations:
no
GLP compliance:
not specified
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
Metabolic activation:
with and without
Metabolic activation system:
Induced S9 mix with Aroclor 1254, from rat or hamster liver cells.
Test concentrations with justification for top dose:
0; 10; 33; 100; 333; 666; 750; 900; 1000; 2000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of the test material

Untreated negative controls:
yes
Remarks:
choline chloride, glycerol, glycine, mannitol and sodium phosphate.
Negative solvent / vehicle controls:
yes
Remarks:
culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene on all strains with rat (1.5 µg/plate) and hamster (0.75 µg/plate) S9; 4-Nitro-o-phenylenediamine on TA98 without S9 (12µg/plate); sodium azide on TA100 and TA1535 without S9 (2.5 µg/plate); 9-aminoacridine on TA1537 (80 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation with S9 mix or buffer
DURATION
- pre-incubation period: 20 min at 37°C
- Exposure duration of incubation: 48 h at 37°C
Evaluation criteria:
Although procedures for the statistical analysis of Salmonella plate test data have been developed [Margolin et al, 1981], they were not incorporated
into the initial data evaluations. The data were evaluated in an ad hoc manner by each testing laboratory and by NTP personnel. Prior to statistical
analysis no formal rules were used; however, a positive response was indicated by a reproducible, dose-related increase, whether it be twofold over
background or not. The matrix of test strains and activation systems used allowed the investigators to detect trends or patterns that might not be
as evident if only one strain and activation system were examined. In addition to the standard "positive" and "negative" categories, there is also
"questionable" (or "inconclusive"). This applied to low-level responses that were not reproducible within the laboratory or to results that showed a
definite trend but with which the investigator did not feel comfortable in making a " +" or " -" decision. It also included tests in which an elevated
revertant colony yield occurred at only a single dose level. After a decision on the mutagenicity of a sample was made, a request to decode the
sample was sent to the repository, and the code was broken.
Statistics:
The data were subsequently evaluated using an analysis based on the models presented by Margolin et al [1981]. As a result of these statistical
analyses, a number of calls were changed from the original "negative" to "equivocal." The statistical analysis did not result in any "positive" or
"equivocal" calls being called "negative".
Because the criteria for "positive" or "questionable" decisions have evolved during the course of the study and because the recent use of statistical
analysis, some of the results differ from the initial evaluations published in the NTP Technical Bulletins (1980 to 1983).
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
yes, slight to toxic for all strains at high concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

 Strain: TA1535

Dose

No Activation
(Negative)

No Activation
(Positive)

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0       

23

2

13

1.8

26

1.2

11

2.6

9

1.8

7

1.2

11

0.9

11

0.9

10       

 

 

20

1.7

 

 

 

 

 

 

6

0.9

 

 

6

0.6

33       

33

1.7

21

2.6

21

4.2

17

1

13

1.3

7

2

10

1

9

2.5

100       

27

2

23

0.7

18

3.8

10

0.7

11

0.6

7

1.2

14

3.5

9

2.7

333       

24

6.4

41

1.5

21

1.8

15

1.8

9

2

8

1.5

10

1.8

6

1.2

666       

 

 

106

3

23s

0.6

14s

1

 

 

7

1.2

 

 

7

1.2

750       

 

 

 

 

18s

1.5

13s

1.9

 

 

 

 

 

 

 

 

900       

 

 

 

 

14s

5.8

8s

4.1

 

 

 

 

 

 

 

 

1000       

T

 

 

 

 

 

 

11s

0.5

 

 

5s

1

 

 

2000       

T

 

 

 

 

 

 

T

 

 

T

 

 

Positive Control

1408

20.8

1446

11.7

2002

67.1

1413

91.8

148

6.8

85

5.9

77

1

53

8.

Strain: TA100

Dose

No Activation
(Equivocal)

No Activation
(Weak Positive)

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0       

97

2.1

82

5

103

11.9

108

6.7

85

6.9

82

6.1

92

13.2

81

6.2

10       

 

 

88

12.9

 

 

 

 

 

 

83

8.6

 

 

70

8.1

33       

108

9.8

91

4.3

99

4.5

103

15.9

91

3.8

75

2.2

98

6.2

74

2.9

100       

124

2

99

9.5

105

8.5

107

7.6

84

6.5

106

8.4

98

4.3

82

8.4

333       

109

5.4

141

2.3

96

4.7

94

8.7

92

5.2

83

0.9

99

9

81

8.9

666       

 

 

246

17.2

101

3.8

114s

12.7

 

 

75

5

 

 

84

3.5

750       

 

 

 

 

105s

8.1

116s

10.1

 

 

 

 

 

 

 

 

900       

 

 

 

 

74s

33.5

62s

27

 

 

 

 

 

 

 

 

1000       

T

 

 

 

 

 

 

69s

9.5

 

 

T

 

 

2000       

T

 

 

 

 

 

 

T

 

 

T

 

 

Positive Control

1919

39.3

2152

99.5

2173

58.7

1590

94.7

1634

59.6

861

16.2

1303

116.4

457

35.9

Strain: TA98

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

19

2.5

18

5.1

29

3.3

23

0.9

20

3.8

22

2.9

10         

 

 

16

3.6

 

 

16

1.7

 

 

21

2.4

33         

14

0.9

17

3.8

27

2.3

22

3.2

22

1.2

24

0.7

100         

17

2.3

20

4.1

29

2.4

22

3

19

4.1

22

3.1

333         

16

1.5

15

3.3

23

0.9

15

1.9

33

1.7

21

1.2

666         

 

 

14

3.3

 

 

21

1.5

 

 

20

1.2

1000         

T

 

 

27s

6.5

 

 

T

 

 

2000         

T

 

 

T

 

 

T

 

 

Positive Control

1174

17.5

1352

55.2

983

53.9

808

31.9

529

73.4

532

18.2

  Strain: TA1537

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

8

1.9

3

0.6

10

1.7

5

0.7

10

1

6

2.6

10         

 

 

5

1.2

 

 

6

0.3

 

 

5

0.6

33         

6

0.7

4

1.2

9

0.3

4

1.2

7

1.5

3

0.3

100         

6

0.9

3

0.9

7

0.7

3

0.7

10

3.7

4

0.6

333         

8

1.3

4

1.2

10

2.3

5

0.7

9

0.9

8

2.5

666         

 

 

5

0.7

 

 

5

1.2

 

 

7

3.7

1000         

T

 

 

8s

1

 

 

9s

2.6

 

 

2000         

T

 

 

T

 

 

T

 

 

Positive Control

383

200.3

518

52.7

206

29.5

105

14.5

73

4.7

58

3.5

Conclusions:

Negative with and without metabolic acivation.
Executive summary:

In a reverse gene mutation assay in bacteria (Haworth, 1983), strains TA98, TA100, TA1535 and TA1537 of S. typhimurium were exposed to paradimethoxybenzene at concentrations of 0 to 2000 µg/plate in the presence and absence of mammalian metabolic activation (rat or hamster liver cells).

 

Paradimethoxybenzene was tested up to cytotoxic concentrations.

The positive controls induced the appropriate responses in the corresponding strains.   There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline  OECD 471 for in vitro mutagenicity bacterial reverse gene mutation data.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 22 feb 1988 to 20 june 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538
Metabolic activation:
with and without
Metabolic activation system:
S9 from rat liver cells, induced with Aroclor 1254
Test concentrations with justification for top dose:
0, 10, 100, 500, 1000 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: usually used in this type of test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details below
Details on test system and experimental conditions:
Positive controls:
- Without metabolic activation: Sodium azide (3 µg/plate) for TA100 and TA1535; 9-Aminoacridine for TA1537; 2-Nitrofluorene (0.5 µg/plate) for TA98 and TA1538.
- With metabolic activation: 2-Aminofluorene (5 µg/plate) for TA98 and TA1538.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- incubation period: 48 hours at 37°C
- Exposure duration: 48 hours

NUMBER OF REPLICATES: 3
Evaluation criteria:
- in TA 98 and TA 100: a test article is considered as positive if it produce at least a 2-fold increase in the mean revertant per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control.
This increase in the number of revertant must be accompanied by a dose response with increasing concentrations of the test article
- in TA 1535, TA 1537 and TA 1538: idem, but a 3-fold increase is required
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Tables of results (2 studies):

 

 

TA 98

TA 100

TA 1535

Conc.

[unit]

-S9

+S9

Cytotoxic

ity

-S9

+S9

Cytotoxic

ity

-S9

+S9

Cytotoxic

ity

0*

25/21

28/28

no

94/94

92

no

15/22

11/14

no

10

25/20

29/28

no

95/85

87

no

13/14

12/10

no

100

20/19

34/27

no

96/98

85

no

21/18

13/15

no

500

18/19

28/32

no

105/96

84

no

18/18

15/12

no

1000

20/24

21/33

no

98/95

93

no

18/27

9/17

no

5000

14/14

10/1/

no

84/83

79

no

15/25

8/9

no

Positive control

182/216

2012/1556

no

1036/889

/

no

938/697

/

 

no

 

 

TA 1537

TA 1538

Conc.

[unit]

-S9

+S9

Cytotoxic

ity

-S9

+S9

Cytotoxic

ity

0*

6/7

7/6

no

10/12

22/21

no

10

7/7

7/6

no

11/11

28/22

no

100

7/5

8/5

no

14/13

25/25

no

500

9/6

7/9

no

8/9

21/24

no

1000

6/6

9/8

no

13/14

21/24

no

5000

7/6

7/4

no

14/13

22/17

no

Positive control

1419/1563

/

no

283/239

1947/1435

no

*solvent control with DMSO

Conclusions:

Negative in Ames test, with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria (Hoechst, 1988), strains TA98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Paradimethoxybenzene (purity unknown), in DMSO at concentrations of 0, 10, 100, 500, 1000 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (plate incorporation method).

Paradimethoxybenzene was tested up to the limit concentration (5000 µg/plate). It was not mutagenic in Ames test.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

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

Additional information

Genotoxicity in vitro:

Two studies were available: one with relaibilty 1 (GLP study, Hoechst, 1988) and another one with reliability 2 (Haworth, 1983). They were selected as key studies.

In these reverse gene mutation assays in bacteria, strains TA98, TA100, TA1535, TA1537 and TA1538 of S. typhimurium were exposed to paradimethoxybenzene at concentrations of 0 to 5000 µg/plate in the presence and absence of mammalian metabolic activation (rat or hamster liver cells).

Paradimethoxybenzene was tested up to cytotoxic concentrations.

The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background in these two studies.

Genotoxicity in vivo:

Only one study (Hoechst, 1996) was available and was selected as key study.

The micronucleus test was performed with Paradimethoxybenzene, according to the OECD 474 guideline under GLP conditions.

The test compound was suspended in starch mucilage and was given once as an orally dose of 2000 mg/kg bw to male and female NMRI mice, based on the results of a preliminary study.

According to the test procedure, the animals were killed 12, 24 or 48 hours after administration.

Endoxan (cyclophosphamide in distilled water) was used as positive control substance and was administered once orally at a dose of 50 mg/kg bw.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Paradimethoxybenzene and was statistically not different from the control values.

Positive control induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

Under the test conditions, the results indicate that Paradimethoxybenzene is not mutagenic in the micronucleus test.


Justification for classification or non-classification