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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
other: the study was performed with the sulfate salt of 1,4-diamino-2-methoxymethylbenzene
Adequacy of study:
key study
Study period:
From Dec. 19, 2006 to Dec. 22, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
according to German and OECD principles of GLP
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
337906-37-3
Cas Number:
337906-37-3
IUPAC Name:
337906-37-3
Constituent 2
Reference substance name:
-
EC Number:
474-270-7
EC Name:
-
IUPAC Name:
474-270-7
Constituent 3
Reference substance name:
1,4-Diamino-2-methoxymethyl-benzene sulfate (1:1)
IUPAC Name:
1,4-Diamino-2-methoxymethyl-benzene sulfate (1:1)
Details on test material:
- Name of test material: 1,4-Diamino-2-methoxymethyl-benzene sulfate (1:1), Methoxymethyl-PPD-Sulfat, WR801337
(Code # A012220)
- TSIN: 801337 
- Molecular formula: C8H12N2O x H2SO4
- Molecular weight: 250.28
- Smiles notation: Not reported
- InChl: Not reported
- Substance type: Pure active substance
- Physical state: Cream-rosy powder
- Stability: The substance on storage in dryness and darkness is considered to be stable for more than 8 years.
- Stability in Solution: 1, 4-diamino-2-methoxymethyl-benzene sulfate (1:1) was considered stable for 7 d in water (approx. 10% solution), DMSO (approx. 10% solution), and water/acetone 1:1 (approx. 1.5% solution) at room temperature
. During the course of storage no decomposition of the test substance could be observed.
- Storage condition of test material: At room temperature
- Solubility: Solubility in different solvents is as follows:  
> 10 weight% in water (pH 1.75);
0.03 weight% in acetone;
1.7 weight% in acetone/water (1:1);
15 weight% in DMSO

Method

Target gene:
Histidine locus serves as a specific target gene of following Salmonella Typhimurium tester strains :

TA 1537: his C 3076 (Frame shift mutation) 
TA98: his D 3052 (Frame shift mutation) 
TA 1535 and TA 100: his G 46 (Base – pair substitution) 
TA 102: his G 428 (Base – pair substitution)

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: (rfa-) mutation (faulty lipopolysaccharide envelope); deletion of the uvrB gene; nitrate reductase and biotin deficient; R-factor plasmid (error prone repair) and ampicillin resistance marker (in TA 98 and TA 100 only)
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: (rfa-) mutation (faulty lipopolysaccharide envelope); R-factor plasmid (error prone repair), multicopy plasmid pAQ1 (hisG428 mutation gene) and tetracycline resistance gene
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment and Main Experiment: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
The pre-experiment was reported as Main experiment since no toxic effects were observed and 5000 µg/plate was chosen as maximal concentration.
Vehicle / solvent:
- Vehicle used: Deionised water 
- Justification for choice of solvent/vehicle: The solvent was chosen based of its solubility properties
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: Prepared in deionized water at a concentration of 10 µg/plate for TA 1535 and TA 100
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD); Prepared in DMSO at a concentration of 10 and 50 µg/plate for TA 98 and TA 1537 respectively
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: Prepared in deionized water at a concentration of 30 µg/plate for TA 102
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene; Prepared in DMSO at a concentration of 2.5 µg/plate for TA 1535, TA 1537, TA 100 and TA 98 and 10.0 µg/plate for TA 102
Details on test system and experimental conditions:
MAINTAINENCE OF TESTER STRAIN: The bacterial tester strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 were obtained from Trinova Biochem GmbH (35394 GieBen, Germany). The strains were precultured and stored as follows:

- Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth containing 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

- Periodic checking: Regular checking of the properties of the strains regarding the membrane permeability, ampicillin and tetracycline resistance as well as spontaneous mutation rates was performed to ensure that the experimental conditions set down by Ames were fulfilled.

- Precultures: From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 µL tetracycline (2 µg/mL) was added to strain TA 102. This nutrient medium contains per liter:
8 g Merck Nutrient Broth (MERCK, D-64293) 
5 g NaCl (MERCK, D-64293)

The bacterial cultures were incubated in a shaking water bath for 4 h at 37°C.

METHOD OF APPLICATION: In agar (direct plate incorporation)

EXPERIMENTAL PROCEDURE: 100 µL test solution at each dose level/solvent control/ negative control/reference mutagen solution (positive control), 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 µL Bacteria suspension (cf. test system, pre-culture of the strains) and 2000 µL overlay agar were mixed in a test tube and poured onto the selective agar plates. After solidification the plates were incubated upside down for at least 48 h at 37°C in the dark.

NUMBER OF REPLICATIONS: 3 plates/strain/dose level

- Method for evaluation: Reduction in the number of spontaneous revertants or clearing of bacterial background lawn

Evaluation criteria:
- The test substance was considered a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100, and TA102) or three times (strains TA1535 and TA1537) the colony count of the corresponding solvent control was observed.

- A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.

- An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.

- A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data was not required.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance occurred up to the highest investigated dose.

PRE- EXPERIMENT TOXICITY RESULTS: In the pre-experiment toxicity study, no toxic effects were observed and evaluable plates (>0 colonies) were obtained at five concentrations or more in all strains tested. Based on these results, pre-experiment was reported as Main Experiment and 5000 µg/plate was chosen as maximal concentration.

MAIN MUTAGENICITY EXPERIMENT RESULTS:
No significant increases in revertant numbers were seen in any of the strains treated in the absence of S9. In the presence of S9, statistically and biologically significant increases in revertant numbers were seen in 4 strains. The maximum responses, all seen at 2500 μg/plate, can be summarised as follows:
• 7.7-fold increase in TA1535 revertants
• 27.0-fold increase in TA1537 revertants
• 27.9-fold increase in TA98 revertants
• 8.5-fold increase in TA100 revertants.
Thus, both base substitution and frameshift mutations were induced, but the highest responses were for frameshift mutations.
For details, refer to "table 1" under "Any other information on results incl. tables".

COMPARISON WITH HISTORICAL CONTROL DATA: Negative control, solvent control and positive control data in this study were comparable with the historical control data (From May 2005 until June 2006)

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without metabolic activation in all strains used.


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Number of revertant colonies in reverse mutation assay of1,4-Diamino-2-methoxymethyl-benzene sulfate (1:1) (Methoxymethyl-PPD-Sulfate) (Study # 58456)

Metabolic Activation

Test Group

Dose Level (µg/plate)

Revertant Colony Counts (Mean±SD)

TA 1535

TA 1537

TA98

TA 100

TA 102

Without Activation

DMSO

 --

23 ± 1

13 ± 7

29 ± 1

115 ± 6

401 ± 34

Untreated

 --

19 ± 2

14 ± 7

25 ± 15

140 ± 4

387 ± 26

Methoxymethyl-PPD-Sulfate

3

21 ± 4

9±2

21 ± 7

127 ± 12

338 ± 14

10

22 ± 2

14 ± 8

25 ± 5

112 ± 12

444 ± 14

33

23 ± 5

13 ± 1

25 ± 3

126 ± 11

437 ± 23

100

24 ± 5

10 ±4

34 ± 6

123 ± 12

435 ± 8

333

16 ± 3

15 ± 3

26 ± 3

115 ± 8

374 ± 42

1000

18 ± 4

11 ± 5

31 ± 6

123 ± 23

421 ± 18

2500

27 ± 7

17 ± 4

36 ± 12

140 ± 14

446 ± 27

5000

22 ± 9

18 ± 4

38 ±9

137 ± 5

454 ± 12

NaN3

10

1785 ± 62

 --

-- 

1989 ± 96

-- 

4-NOPD

10

-- 

 --

368 ± 24

-- 

-- 

4-NOPD

50

-- 

90 ± 3

-- 

-- 

 --

MMS

3µL

-- 

 --

 --

-- 

4767 ± 275

With Activation

DMSO

-- 

26 ± 1

16 ± 4

45 ± 7

142 ± 14

541 ± 10

Untreated

-- 

19 ± 4

13 ± 5

41 ± 5

129 ± 6

480 ± 38

Methoxymethyl-PPD-Sulfate

3

16 ± 3

21 ± 3

43 ± 2

126 ± 24

449 ± 35

10

17 ± 5

22 ± 2

57 ± 9

148 ± 19

431 ± 11

33

24 ± 9

22 ± 2

67 ± 9

177 ± 28

527 ± 26

100

34 ± 6

38 ± 1

119 ± 10

188 ± 5

494 ± 72

333

72 ± 6

89 ± 6

419±10

260 ± 24

426 ± 45

1000

157 ± 18

253 ± 11

1072 ± 64

646 ± 23

557 ± 23

2500

201 ± 29

432 ± 5

1258 ± 194

1208 ± 68

571 ± 98

5000

156 ± 13

406 ± 56

737 ± 9

901 ± 9

509 ± 87

2-AA

2.5

319±13

207 ± 57

1198 ± 224

1855 ± 132

-- 

2-AA

10.0

 --

 --

 --

-- 

1792 ± 185

NaN3: Sodium azide

2-AA: 2-aminoanthracene

MMS: methyl methane sulfonate

4-NOPD: 4-nitro-o-phenylene-diamine

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100 in the presence but not in the absence of metabolic activation. Therefore, 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay in the presence of S9.
Executive summary:

The bacterial reverse mutation test of 1,4-Diamino-2-methoxymethyl-benzene sulfate (Methoxymethyl-PPD-Sulfat) was determined following OECD guideline 471 (Bacterial Reverse Mutation Test.).

2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I and II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed with and without liver microsomal (S9 mix) activation. Each concentration, including the controls, was tested in triplicate. The test susbtance was tested at the following concentrations: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No significant increases in revertant numbers were seen in any of the strains treated in the absence of S9. In the presence of S9, statistically and biologically significant increases in revertant numbers were seen in 4 strains. The maximum responses, all seen at 2500 μg/plate, can be summarised as follows:

• 7.7-fold increase in TA1535 revertants

• 27.0-fold increase in TA1537 revertants

• 27.9-fold increase in TA98 revertants

• 8.5-fold increase in TA100 revertants.

Thus, both base substitution and frameshift mutations were induced, but the highest responses were for frameshift mutations.Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100 in the presence but not in the absence of metabolic activation. Therefore, 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay in the presence of S9.

This bacterial reverse mutation test is classified as acceptable, and satisfies the guideline requirements of the OECD 471 method.

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