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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
2-methyl-p-phenylenediamine sulphate when administered orally at 20, 40 and 80 mg/kg bw did not induce unscheduled DNA synthesis in primary rat hepatocytes in autoradiographic in vivo unscheduled DNA synthesis assay.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From Apr. 26, 2002 to July 23, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Reference:
Composition 0
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
GLP compliance:
yes
Remarks:
according to US FDA, OECD and Italian legislation principles of GLP
Type of assay:
unscheduled DNA synthesis
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Harlan Italy S.r.l., San Petro al Natisone (UD), Italy- Age at study initiation: 7-10 wks- Weight at study initiation: 200-300 g- Assigned to test groups randomly: On the day of allocation rats were assigned to the treatment groups and cages. Animals were uniquely identified by ear notch and tail marks, and housed 4 per cage.- Fasting period before study: 10-15 h prior to treatment- Housing: On arrival the animals were examined for overt signs of ill health and housed up to 5 to a cage during acclimation. The animals were housed in clear polycarbonate cages measuring 59x39x20 cm with a stainless steel mesh lid and floor. Each cage held absorbent paper which was inspected daily and changed as necessary. On the day of allocation the animals were assigned to groups and housed 4 per cage.- Identification of animals: The cages were identified by a label, colour-coded according to groups and recording the study number, animal numbers and details of treatment. Animals from the main experiments were weighed on the day of treatment.- Diet: ad libitum, fixed formula rodent diet (Altromin MT diet)- Water: ad libitum- Acclimation period: Minimum of 5 dENVIRONMENTAL CONDITIONS- Temperature: Animal room controls were set to maintain temperatures at 22± 2°C. Actual conditions were continuously monitored and recorded.- Humidity: Animal room controls were set to maintain relative humidity at 55±15%. Actual conditions were continuously monitored and recorded.- Photoperiod : 12 h dark /12 h lightEXPERIMENT INITIATION DATE: Apr. 26, 2002EXPERIMENT COMPLETION DATE: July 23, 2002
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Sterile injectable water - Justification for choice of solvent/vehicle: Not reported- Amount of vehicle: 10 mL/kg bw- Lot/batch no. (if required): Bieffe Medital; Batch # 01C0201
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Solutions of the test substance were prepared immediately before use in the vehicle.
Duration of treatment / exposure:
14 h
Frequency of treatment:
Single administration
Post exposure period:
Experiment 1: 2 hExperiment 2: 14 h
Remarks:
Doses / Concentrations:20, 40 and 80 mg/kg bw Basis:actual ingested
No. of animals per sex per dose:
4 males/group. One animal from each dose group was held in reserve.
Control animals:
yes, concurrent vehicle
other: positive control
Positive control(s):
Experiment 1 (2 h sampling time):The solution of methylnitrosourea (MNU) prepared in sterile injectable water - Justification for choice of positive control(s): Not reported- Route of administration: Oral gavage- Lot/Batch No.: Sigma Chemical Co.; Batch # 37H1158- Doses / concentrations: 80 mg/kg (Concentration: 8 mg/mL) - Dose volume: 10 mL/kg Experiment 2 (14 h sampling time): Solutions of 2-acetylaminofluorene (2-AAF) prepared in corn oil/DMSO mixture where the DMSO concentration did not exceed 2%.- Justification for choice of positive control(s): Not reported- Route of administration: Oral gavage- Lot/Batch No.: Sigma Chemical Co.; Batch # 40H0138- Doses / concentrations: 100 mg/kg (Concentration: 12.5 mg/mL)- Dose volume: 8 mL/kg
Tissues and cell types examined:
Hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels selected were based on information supplied by the sponsor.TREATMENT AND SAMPLING TIMES: Two experiments, using the auto radiographic method to determine UDS, were performed.The animals were treated once with vehicle/test solution/positive control and were observed for clinical signs at approx. 30 min after dosing and before sacrifice for Experiment 2. Animals were sacrificed and hepatocytes were extracted at approx. 2 (Experiment 1) or 14 (Experiment 2) h after treatment in both studies. DETAILS OF SLIDE PREPARATION (Preparation of hepatocyte cultures and autoradiography): The rats were anaesthetized by the intraperitoneal administration of Sodium Pentothal at a dose of approx. 80 mg/kg bw. Primary rat hepatocytes were prepared by perfusing rat livers in situ. Liver perfusions were undertaken for 3 animals per group approx. 2 h after treatment in the Experiment 1 and approx. 14 h after treatment in the Experiment 2. The cells were plated and allowed to attach for at least 90 min. Cell cultures were then treated with tritiated thymidine for a period of at least 4 h and further after washing with PBS were mounted on microscope slides. The slides were dipped briefly into molten autoradiographic emulsion (Kodak NTB2) for autoradiographic measurement of UDS (Unscheduled DNA Synthesis). After an exposure period of 14 d the slides were developed and stained.Details on perfusion of rat liver are provided in the study report.- Number of replicates: Six replicate cultures were prepared at each dose level. METHOD OF ANALYSIS (Net grain count): Counting of nuclear and cytoplasmic grains was performed by microcopy using a high resolution objective under oil immersion.Each slide was examined to ensure that the treated culture had remained viable; for one animal in the low dose group of experiment 2 only one replicate was considered suitable. Two slides with 50 cells were scored for each animal in order to obtain a total of 100 cells, with the exception of the one animal in the low dose group described above where counting was performed on one slide. The cytoplasmic count was subtracted from the nuclear media count to give net grains/nucleus (NG). The percentage of cells in repair (defined as cells with a net grain count of at least +5) was calculated for each animal. In both experiments, slides generated at 20 and 40 mg/kg bw were selected for scoring.
Evaluation criteria:
The assay was considered valid if the following criteria were met:- Vehicle control animal had a group nuclear net grain value within or contiguous with the historical control range (-8.02 to -0.10);- Positive control animals had a group mean nuclear net grain value of five or greater.In the assay the test substance was considered to induce UDS if the following criteria were met:- A minimum net grain count of 0 grains per nucleus was consistently observed in more than one treated animal.- At least 20% of the cells were in repairExamination of the percentage of cells in repair (mean NG>/=5) ensured that any increase in labeling was general and that increased net grain count values were not the results of a few highly labeled cells.Evidence of a dose-response relationship was considered as supportive evidence for genotoxic activity but was not a requirement for a positive result.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 80 mg/kg bw for the 14 h sampling time
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RESULTS OF UNSCHEDULED DNA SYNTHESIS- Clinical signs/mortality: Following treatment with the test substance, mortality was observed in animals dosed at 80 mg/kg bw for the 14 h sampling time. No cytotoxic effects were observed in hepatocyte preparations at 20 and 40 mg/kg bw. Therefore only cells from animals dosed with 20 or 40 mg/kg-bw were selected for scoring.- Net gram per nucleus: Treatment with test substance at doses up to 40 mg/kg bw yielded NG (net gram) values less than zero, producing group mean NG values over the two experiments in the range -1.26 to -0.09. No cells were seen in repair in any treated culture at this dose level.RESULTS OF SOLVENT AND POSITIVE CONTROL GROUP:- The mean NG of animals from the vehicle control group was in the range of-1.98 to -1.84 (Experiment I) and of -0.93 to -0.83 (Experiment II). These values were within the historical control range.- The mean NG of the positive control was in the range of 4.76 to 5.98 (2-AAF) and 4.48 to 5.91 (MNU). The percentage of cells in repair was in the range of 55% to 72% (2-AAF) and 65% to 80% (MNU). Marked increases in UDS were observed following treatment with the positive control substance, indicating the correct functioning of the test system.

Table 1: Summary of UDS in primary rat hepatocytes after in vivo treatment (Experiment 1 (sampling time: 2 h) (study # 71361 and 71580)

Animal Number

Dosage (mg/kg )

NG ± SD (mean/animal)

NG (mean/group) ± SE

% cells in repair/animal

% cells in repair/group ± SE

98

0

-1. 84 ± 1.37

-1.91 ± 0.04

0.0

0.0 ± 0.0

100

-1.98 ± 1.15

0.0

102

-1. 90 ± 1.34

0.0

106

20

-1. 40 ± 1.06

-1. 09 ± 0.42

0.0

0.0 ± 0.0

108

-1. 60 ± 1.34

0.0

110

-0.26 ± 0.63

0.0

114

40

-0.18 ± 0.63

-0.09 ± 0.06

0.0

0.0 ± 0.0

116

-0.11 ± 0.30

0.0

118

0.03 ± 0.37

0.0

Table 2: Summary of UDS in primary rat hepatocytes after in vivo treatment (Experiment 2 (sampling time: 14 h)) (study # 71361 and 71580)

Animal Number

Dosage (mg/kg )

NG ± SD (mean/animal)

NG (mean/group) ± SE

% cells in repair/animal

% cells in repair/group ± SE

138

0

-0.83 ± 0.85

-0.88 ± 0.03

0.0

0.0 ± 0.0

140

-0.93 ± 0.82

0.0

142

-0.89 ± 0.95

0.0

146

20

-0.81 ± 0.84

-0.86 ± 0.02

0.0

0.0 ± 0.0

148

-0.88 ± 0.73

0.0

150

-0.88 ± 0.76

0.0

154

40

-1.21 ± 0.94

-1.26 ± 0.07

0.0

0.0 ± 0.0

156

-1.18 ± 0.94

0.0

158

-1.40 ± 1.26

0.0

In study 9622A, the slides from the 80 mg/kg bw dose group that were not initially evaluated during the performance of study 9622 were reviewed and evaluated. It was identified that a dose of 80 mg/kg bw there was a group mean NG value of zero observed. However, an increase in the mean NG count in one animal was observed. It was determined that the increase was a not the result of an increase in nuclear labeling, but rather due to a depressed cytoplasm count. Hence this result was not ascribed to the genotoxic action of the test substance, but attributable to a cytotoxic effect. No cells in repair were observed in any animal.

Conclusions:
Interpretation of results (migrated information): negative2-methyl-p-phenylenediamine sulphate (SAT 010935) when administered orally at 20, 40 and 80 mg/kg bw did not induce unscheduled DNA synthesis in primary rat hepatocytes in autoradiographic in vivo unscheduled DNA synthesis assay.
Executive summary:

Thein-vivo DNA damage and repair assay of 2-methyl-p-phenylenediamine sulphate (SAT 010935) was determined following OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo).

Male Sprague-Dawley SD rats weighing200-300 gfromHarlan Italy S.r.l., San Petro al Natisone (UD), Italywere acclimated for minimum of 5 d for this study. The food used wasfixed formula rodent diet(ad libitum). The animals were housed 5/cage inclear polycarbonate cages measuring 59x39x20 cm with a stainless steel mesh lid and floorand maintained under standard laboratory conditions (temperature: 22 ±2°C, humidity: 55±15%; artificial light: 12-h cycle). 

The induction of UDS was evaluated using the definitive autoradiographic method. The test substance was formulated in sterile injectable water, which was also used as vehicle control and was administered once by oral gavage. The volume administered orally was 10 mL/kg bw.

 Two independent experiments with two sampling time (2 and 14 h) were performed in this study.

The following dose levels with 4 male per dose groups were investigated:

Experiment 1 (2 h sampling time):20, 40 and 80 mg/kg bw 

Experiment 2 (14 h sampling time):20, 40 and 80 mg/kg bw

Methylnitrosourea(80 mg/kg bw) and2-acetylaminofluorene (2-AAF at 100 mg/kg bw), was used as positive control in Experiment 1 and Experiment 2 respectively.

Liver perfusions were undertaken for 3 animals per group approx. 2 h after treatment in the first experiment and approx. 14 h after treatment in the second experiment. Primary rat hepatocytes were prepared by perfusing rat livers in situ. The cells were plated and allowed to attach for at least 90 min. The cell cultures were treated with tritiated thymidine for a period of at least 4 h for autoradiographic measurement of UDS after treatment with tritiated thymidine.

Six replicate cultures were prepared at each dose-level. After an exposure period of 14 d the slides were developed and stained. In both experiments, slides generated at 20 and 40 mg/kg bw were selected for scoring. Two slides (100 cells) from each animal were analysed. Nuclear and cytoplasmic grains were

counted separately and the cytoplasmic counts were subtracted from the nuclear counts to give net grains/nucleus (NG).

At 80 mg/kg bw mortality was observed in animals after the administration of test material. Treatment with the test substance at 20 and 40mg/kgbody weight did not produce a group mean net grain value greater than -0.09, no cells were found in repair at either dose.

Based on above, 2-methyl-p-phenylenediamine sulphate (SAT 010935) when administered orally at 20, 40 and 80 mg/kg bw did not induce unscheduled DNA synthesis in primary rat hepatocytes in autoradiographicin vivo unscheduled DNA synthesis assay.

Thisin-vivo DNA damage and repair assay is classified as acceptable, and satisfies the guideline requirements of the OECD 486 method.

 

 

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

Additional information

Additional information from genetic toxicity in vivo:

Justification for selection of genetic toxicity endpoint
more recent data available on similar substance used for read across 2-methyl-p-phenylenediamine sulphate- reliability 2

Justification for classification or non-classification

In vitro and in vivo studies were available on 2 -methyl-p-phenylenediamine and 2 -methyl-p-phenylenediamine sulphate. Both substances present idenitical effects and data on 2-methyl-p-phenylenediamine could be used as reference for evaluation of genotoxic effects.

Some in vitro mutagenic and genotoxic studies presented positive results but the in vivo valid studies indicated no mutagnic or genotoxic effects with the substance, which is therefore considered as no mutagenic.