1,4-Benzenediamine, 2-(methoxymethyl)-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

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 Sept. 01, 2003 to Oct. 10, 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets basic scientific principles, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Comet assay was preformed to detect DNA single strand break representing a primary DNA damage, which may result in mutations or may be repaired. Increase in tail length (parameter to measure genotoxicity) of liver, stomach and urinary bladder epithelium cells were observed in treated and vehicle control animals. Increase in tail length value indicates the DNA damage. Comparison between treated and control animals were performed to determine ability of test substance to cause genetic toxicity.
The conduct and evaluation of the study was performed according to an internationally accepted protocol for the Comet Assay in vivo ( Hartmann, A.; Agurell, E.; Beevers, C.; Brendler-Schwaab, .; Burlinson, B.; Clay, P.; Collins, A.; Smith, A.; Speit, G.; Thybaud, V.; Tice, R. R.: Recommendations for conducting the in vivo alkaline Comet assay; Mutagenesis: Vol. 18, 45-51, 2003)

GLP compliance:

yes

Remarks:

OECD and German principles of GLP

Test material

Test material information

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: C6H12N2O × H2SO4
- Molecular weight: 250.28
- Substance type: Pure active substance 
- Physical state: Cream-rosy powder 
- Stability under test conditions: Test substance on storage in dryness and darkness was considered to be stable for more than 3 yrs.
- Solubility: Solubility of test substance in different solvents is a follows: 
Water: > 10weight% (pH=1.75) 
Acetone: 0.03 weight%
Acetone/ water (1:1): 1.7 weight% 
DMSO: > 10 weight% 
- Stability in solution: Test substance was stable in water (10% w/v, pH: 5.1) in presence of ascorbic acid for 7 d when stored at ambient temperature in absence of light.
- Storage condition of test material:
Test substance was stored at room temperature.

Test animals

Species:

rat

Strain:

other: CRL:(WI) BR Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS 
- Source: Charles River, Sulzfeld, Germany 
- Age at study initiation: 6-8 wks 
- Weight at study initiation: 152-193 g (on the day treatment)
- Assigned to test groups randomly: Yes, animals were randomized according to randomization numbers generated by an in-house created and validated computer program. 
- Fasting period before study: Animals were fasted 4-5 h prior to first treatment. Animals received food again about 1 h after the first treatment. 
- Housing: Animals were housed individually in Makrolon® type II cages with soft wood granules; type S 8/15 (J. Rettenmaier & S5hne, Fillistoff-Fabriken, 73494 Ellwangen-Holzmilhle, Germany) type of bedding. The wood granules were spot-checked for contaminants at regular intervals. 
- Diet: The fixed formula feed "Mliuse/Ratten-Erhaltungsfutter 9441 15 W10" for rats, ad libitum.
- Water: Tap water, ad libitum
- Acclimation period: At least 5 d prior to treatment
- The nutritive composition and contaminant content of the standard diet were routinely spot-checked and analyzed.


ENVIRONMENTAL CONDITIONS
- Temperature: 22.0-22.5°C (required range: 22±1.5°C) 
- Humidity: 43-55% (required range: 40-70%) 
- Air changes: 10 times/ h
- Photoperiod: 12 h of electrical lighting daily

EXPERIMENT STARTING DATE: Sep. 01, 2003
EXPERIMENT COMPLETION DATE: Oct. 10, 2003

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Deionized water with 0.4% ascorbic acid and adjusted to pH 5.1 with NaOH

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in deionized water with 0.4% ascorbic acid and adjusted to pH 5.1 with NaOH. The formulation was freshly prepared for each treatment.
 
PROCEDURE FOR DOSING: For each animal the respective amount of test substance was drawn up in a syringe and was orally administered by stomach tube.
 
DOSE VOLUME: 20 mL/kg bw for all test substance treatment groups

Duration of treatment / exposure:

23 h

Frequency of treatment:

Test substance and negative control: Twice with same dose. Animals were treated for second time after 20 h of first treatment.

Positive control: Once

Post exposure period:

3 h after the second dose

Doses / concentrations

Remarks:

Doses / Concentrations:
25, 50 and 100 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Each treatment group comprised of 5-7 assessable animals/group as follows:
Vehicle control: 6 animals
Each test group: 5 animals
Positive control: 7 animals

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Ethylmethanesulfonate
- Preparation of positive control: The positive control EMS was suspended in corn oil. 
- Application volume: 10 mL/kg bw
- Justification for choice of positive control(s): Not reported
- Route of administration: Oral (gavage) 
- Dose: Once with 300 mg/kg bw

Examinations

Tissues and cell types examined:

Cell from three organs liver, stomach and urinary bladder epithelium cells were analyzed from the dosed animals after necropsy.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses were selected based upon two pilot study performed at various dose concentrations. Based on the result of pilot studies, 100 mg/kg bw (maximum tolerated dose) of test substance and 300 mg/ kg bw dose of positive control was selected for main study.
Further details on pilot study are provided in “Any other information on materials and methods incl. tables”.

COMET ASSAY
- Treatment and sampling times: Animals were treated with test substance and again treated with same dose for second time after 20 h of first treatment.  
- Sacrifice: All test substance treated and vehicle treated animals were sacrificed 23 h after first treatment. Positive control animals were sacrificed 3 h after treatment. Animals were sacrificed by treatment with I.P. injection with Narcoren (Pentobarbital-sodium; 32-48 mg/ animal).
- Procedure for liver perfusion in-situ: Animals were anesthetized and liver was perfused with perfusion solution I and II. After perfusion, the liver was isolated and taken in sterile hood to prepare cells. The details on liver perfusion are provided in study report.
- Preparation of hepatocytes: After perfusion, primary hepatocytes were prepared according to the protocol of Butterworth et al. (1987) under sterile conditions. Briefly, incisions were made in each of the liver lobes and liver cells were carefully collected with the help of a metal comb. The obtained cell suspension was filtered over gross gauze, filled up to 50 mL with cold WEI (Williams Medium E with 1% L-glutamine, 0.1% gentamycin sulfate, without fetal calf serum) and kept on ice for 5-10 min. Afterwards, the resulting supernatant was discarded and the pellet resuspended in 50 mL of cold WEI. The cell suspension was again filtered, this time over fine gauze, and was filled up to 50 mL with WEI. Cells were centrifuged at 50xg for 3 min (<15°C).Cell pellet was resuspended in WEI and again centrifuged with earlier conditions. The resulting pellet was resuspended in 25 mL WEI.
- Preparation of stomach and urinary bladder epithelium cells: The stomach and urinary bladder was removed and transferred to a Petri dish containing cold HBSS. Stomach and urinary bladder was incubated with stirring HBSS buffer (containing a 0.25% trypsin and 0.1% EDTA) in water bath for 25-30 min (for stomach) and 70 min (for urinary bladder) at 37°C. Digestion of stomach was stopped by adding 0.75 mL fetal calf serum (FCS). The resulting cell suspension was centrifuged at approx. 47xg and 4°C for 10 min. The cell pellet was resuspended with 15 mL HBSS.
- Determination of cell viability: After completion of incubation, aliquots of different cells were taken for the determination of cell viability. Cell viability was determined by trypan blue exclusion method. The obtained viability value of the cell suspension after perfusion was a measure for the substance induced cytotoxicity during in vivo exposure. Isolated cells were subsequently subjected to the comet assay procedure.
 
PROCEDURE FOR COMET ASSAY:
- The comet assay was performed according to Singh et al. (1988) with minor modifications. Aliquots of the liver, stomach and urinary bladder epithelium cell suspensions were taken to reach an approx. viable cell number of 4-5 x10000 cells, respectively.
- Cells were centrifuged at about 100xg for 5 min and pellet was mixed with 50 µL of low melting agarose (LMA) (0.7%). The cell/ LMA suspension was carefully pipetted onto slides (76 X 26 mm, ground on one side) already covered with two layers of 0.5% normal melting agarose (NMA; 50 µL each).
- Afterwards, a second LMA layer (0.7%, 50 µL) was placed on top (agarose from Biozym). Starting with the lysis all subsequent steps were performed under red light. After lysis (overnight; lysis buffer containing Na2EDTA, sarcosinate, and freshly added Triton X-100 and DMSO), alkaline treatment (20 min, NaOH and Na2EDTA, pH ≥ 13, in an ice bath) was performed followed by electrophoresis in an ice bath and neutralization (Tris base, pH 7.5).
- Electrophoresis conditions were 40 min, 25 V, 300 mA, for the liver and urinary bladder epithelium cells. Electrophoresis of stomach cells was performed under the same conditions, but for 30 min.
 
CELL QUALITY CRITERIA:
- The viability of liver, stomach and urinary bladder epithelium cells of the vehicle control animals collected by this process should normally exceed 70%.
- For single animals values between 50% and 70% viability may also be accepted, if the cell preparation demonstrates adequate quality.
- Vehicle control animals with organ cell preparations below 50% viability are considered unacceptable. No such limits are set for treated animals.
 
DETAILS OF SLIDE PREPARATION: Subsequently, slides of liver, stomach and urinary bladder cells were stained with ethidium bromide.

No other details on slide preparation are provided in study report.

METHOD OF ANALYSIS: Evaluation of comet assay was performed using image analysis and a validated evaluation program (Comet III, Perceptive Instruments, Haverhill, UK). Tail length, defined as distance between the middle of the head and the end of the tail, was used as assessment parameter.

NUMBER OF CELLS SCORED: 50 cells/slide and two slides/animal were scored (100 cells total).

Evaluation criteria:

The criteria used for assessment of the data are as follows:
- A response is considered positive, if a chemical induces a dose dependent mean increase of the tail length/ dose group of more than 25% above the vehicle control group mean.
- Mean tail length increases in test compound treated groups between 15% and 25% compared to the vehicle control group have to be considered case by case.
- Increases below 15% compared to the vehicle control group are considered negative. However, these criteria may be overruled by good scientific judgment.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

other: equivocally genotoxic in liver cells

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Sex:

male

Genotoxicity:

negative

Remarks:

in rat stomach and urinary bladder epithelium cells

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

LIVER CELLS:  
- 2 out of 5 animals revealed increased tail length values at a dose of 100 mg/ kg (animal 1: 30.03 µm; animal 2: 32.00 µm) and 50 mg/ kg (animal 1: 35.51 µm; animal 3: 30.20 µm) and 3 out of 5 animals revealed increased tail length values at a dose of 25 mg/kg (animal 3: 31.96 µm; animal 4: 33.93 µm, animal 5: 35.02 µm).
- No biologically relevant increase of the tail length value was induced in the other three or two animals of the respective treatment group compared to the vehicle control animals. 
- The mean tail length values/ dose group revealed a reverse dose-dependency in the liver cells.
- At doses of 100 mg/kg and 50 mg/kg, a 25% increase above the mean tail length value of the vehicle control group according to the assessment criteria for a positive response was not reached. Only in the lowest dose group the mean tail length value/ dose group exceeded the criteria for a positive response (25% increases above the mean vehicle control value).
- Due to the lack of a dose dependency (an inverted dose-response was observed), the relatively small increase in tail length, and the fact that there was no consistent effect within the dose groups (a similar proportion of responding animals in each dose group), the result for the liver was considered to be of questionable biological relevance.
 
STOMACH AND URINARY BLADDER CELLS: 
- In treated animals no biologically relevant increases in tail length values were detected. 
- One animal of the 100 mg/kg group revealed a slightly increased tail length value in the stomach cell (animal 4: 29.33 µm). However, all other animals of the same group had no increased tail length values. Therefore, this increase was considered to be of no biological relevance.
- The mean tail length values/ dose group for both, stomach and urinary bladder epithelium, revealed no increase of the mean tail length compared to the mean tail length of the vehicle control group.

Any other information on results incl. tables

Animal distribution/ dose group: The organs collected from number of rats/ dose group are as follows:

Liver: 5 rats (vehicle control and test substance treatment group) and 6 rats (positive control)

Stomach: 5 rats (from all treatment groups)

Urinary bladder epithelium: 6 rats (vehicle control), 5 rats (test substance treated) and 7 rats (positive control)

OTHER OBSERVATIONS:

Clinical observation: All animals treated with 50 and 100 mg/kg bw test article did show ruffled fur and pallor, as well as a red discolouration of the urine, indicating a good bioavailability of the test material.

Mortality: No mortality was observed in any of the treated animals.

Cytotoxicity: Cells of vehicle controls used for assessment revealed good cell viabilities fulfilling our cell quality criteria. No relevant cytotoxic effects could be observed in liver, stomach and urinary bladder epithelium cells of rats exposed to test substance. The same was true for cells of all evaluated organs of the positive control animals.

RESULT OF POSITIVE CONTROL: Roughened fur, pallor was observed in positive control animals. Clear increases in tail length above the acceptance criteria for the positive control (≥ 30% increase above the mean vehicle control value) were observed in liver, stomach and urinary bladder epithelium of all positive control treated rats used for assessment at a sacrifice time of 3 h after treatment. The increase in tail length after treatment is as follows:

Table 1. Mean Tail Length/ Dose Group of The Rat Comet Assay In Vivo after treatment with Methoxymethyl PPD Sulfate (Study # T 4072906)

Dose Group	Liver (mean tail length ± SD)	Stomach (mean tail length ± SD)	Urinary bladder epithelium (mean tail length ± SD)
Vehicle control	22.66 ± 1.8	23.4 ± 2.6	17.79 ± 1.8
25 mg/ kg bw of test substance	30.07 ± 5.2	22.15 ± 2.3	18.00 ± 2.3
50 mg/ kg bw of test substance	28.00 ± 4.8	24.11 ± 1.8	18.56 ± 1.9
100 mg/ kg bw of test substance	25.71 ± 5.0	23.03 ± 3.8	17.98 ± 2.5
Positive control	*40.22 ± 4.5	38.88 ± 1.1	**26.52 ± 2.8

*= Mean of 6 animals

**= Mean of 7 animals

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: equivocally genotoxic in liver cells and non-genotoxic to rat stomach and urinary bladder epithelium cells
Based on these results and under the conditions described, 2-METHOXY-METHYL-PPHENYLENEDIAMINE SULFATE was considered to produce equivocal results in the comet assay in vivo in rat liver cells. 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE was considered to be non-genotoxic in the comet assay in vivo in rat stomach and urinary bladder epithelium cells.

Executive summary:

The aim of this study was to assess the potential genotoxicity of 2-METHOXY-METHYL-PPHENYLENEDIAMINE SULFATE in the Comet assay in liver, stomach and urinary bladder epithelial cells after in vivo treatment of male rats. The test material was dissolved in deionised water and was administered via gavage (dosing volume 20 mL/kg bw) twice to 5 male Wistar rats at doses of 0, 25, 50 and 100 mg/kg bw/day with a time difference of 20 hours between both administrations. Animals were sacrificed 3 hours after the second administration. Cells of the liver, stomach and urinary bladder epithelium were investigated in this Comet assay.

The selection of the 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE doses was based on two pilot studies. In the first pilot study three male rats were orally administered 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE at a dose of 2000 mg/kg bw. All animals died shortly after application. In a second pilot study, three male rats received 200 mg/kg bw. and four male rats were treated with 100 mg/kg bw at the first application. The application volume was 20 mL/kg. At 200 mg/kg bw. two animals died within 19 hours after treatment. The surviving animal was sacrificed before the second treatment due to the fact that the MTD was exceeded. At 100 mg/kg bw one hour after this dose and before a second dose was administered the following symptoms were recorded: reduced motility, roughened fur, discoloured urine (reddish). A second dose of 100 mg/kg bw was administered 20 hrs following the first dose. Directly after the second dose the following symptoms were observed: roughened fur, pallor, hunched posture. Twenty hours after the second dose discoloured urine (brownish) was observed. Forty eight hours after the second dose all animals showed increased intake of water and frequent micturition. None of the animals died.

The positive control group (4 male rats) received 300 mg/kg bw ethyl methane sulfonate (EMS) in deionised water once via gavage. Two animals were sacrificed 2 hours after treatment and two animals were sacrificed 3 hours after treatment. Portions of the liver, stomach, and urinary bladder were removed, processed, and analyzed for DNA damage using the comet assay. For each comet sample analyzed, 100 cells (50 cell per

slide, if possible) were scored for DNA migration. In addition, 100 cells were assessed for cytotoxicity using the trypan blue dye exclusion test.

All animals treated with 50 and 100 mg/kg bw test material did show ruffled fur and pallor, as well as a red discolouration of the urine, indicating a good bioavailability of the test material.

Slight increases in the mean tail length were detected in the livers of 2-3 out of 5 rats in each of the dose groups after oral treatment with 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE (see table 1 below for group mean + SD values). Due to the lack of a dosedependency

(an inverted dose-response was observed), the relatively small increase in tail length, and the fact that there was no consistent effect within the dose groups (a similar proportion of responding animals in each dose group), the result for the liver was considered to be of questionable biological relevance.

No biologically relevant increase of the tail length value was observed in cells of the stomach and the urinary bladder epithelium after oral treatment of rats with 2-METHOXY-METHYL-PPHENYLENEDIAMINE SULFATE.

The tail length of positive control rats was markedly increased when compared to the negative control animals for liver, stomach and urinary bladder epithelium cells, demonstrating the sensitivity of the test system for the detection of genotoxic effects.

Based on these results and under the conditions described, 2-METHOXY-METHYL-PPHENYLENEDIAMINE SULFATE was considered to produce equivocal results in the comet assay in vivo in rat liver cells. 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE was considered to be non-genotoxic in the comet assay in vivo in rat stomach and urinary bladder epithelium cells.