p-phenylenediamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

- Purity: 99.8%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation at start of treatment:
males mean value 214.6 g (SD ± 10.48 g)
females mean value 172.3 g (SD ± 8.47 g)
- Fasting period before study: not reported
- Housing: singly in Makrolon Type II, with wire mesh top with granulated soft wood bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-80 %
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle Control
- Name: deionised water
- Route and Frequency of Administration: orally, once
- Volume Administered: 10 mL/kg b.w.

Details on exposure:

PREPARATION OF TEST SUBSTANCE FORMULATION:

-Solvent used: deionised water
-Preparation frequency: once
-Preparation details: Before preparation, the vehicle was degassed by sonication for at least 15 minutes and then saturated with nitrogen gas, and kept under nitrogen atmosphere for 15 minutes prior to dosing. The test substance dosage forms were prepared under nitrogen atmosphere and were stored prior touse protected from light and under nitrogen atmosphere until delivery, for a maximum period of 5 hours.

Duration of treatment / exposure:

single oral dose

Frequency of treatment:

single oral dose

Post exposure period:

24 and 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
25, 50, 100 mg/kg b.w.
Basis:
nominal in water

No. of animals per sex per dose:

6 per sex at each dose level: negative control, low dose, medium dose, high dose and positive control

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive Control
- Name: CPA; Cyclophosphamide
- Supplier: Sigma-Aldrich Vertriebs GmbH
- Catalogue no.: C 0768 (purity: > 98 %)
- Justification for choice of positive control(s): not reported
- Dissolved in: deionised water
- Dosing: 40 mg/kg b.w.
- Route and frequency of administration: orally, once
- Volume administered: 10 mL/kg b.w.
- Solution prepared on day of administration. The stability of CPA at room temperature is sufficient. At 25°C only 3.5 % of its potency is lost after 24 hours.

Examinations

Tissues and cell types examined:

PCEs and NCEs from rat bone marrow taken from the femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available. Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 hours after treatment. For the highest dose level an additional sample was taken at 48 hours after treatment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1, 2-4, 6, and 24 hours after administration of the test item. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION:
The nucleated cells were separated from the erythrocytes using the method of Romagna. Briefly, the cell suspensions were passed through a column consisting of α-Cellulose (Sigma) and Cellulose (Sigmacell type 50). The columns were then washed with Hank’s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. The pellet was resuspended in a small drop of FCS and spread on slides. The smear was air-dried and then one slide per animal was stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide per animal was stained with acridine orange.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei using May-Grünwald/Giemsa and acridine orange stained slides. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining 6th animal of each group and sex was evaluated in case an animal of that group died.

OTHER:
The analysis of the test item concentration in blood serum was performed at RCC-ECP under the phase number A26774. For this purpose 3 animals per sex and sampling time were treated at RCC Ltd with radiolabelled test item using the dose corresponding to the high dose of the mutagenicity experiment, i.e. 100 mg/kg. The test item was formulated in degassed deionised water. The volume was as for the mutagenicity experiment 10 mL/kg b.w. 0.5 and 2 hours after the treatment approx. 2 mL of blood was collected via the jugular vein into glass tubes and stored at –60 to -80 °C. Radioactivity in blood was determined by liquid scintillation counting (LSC).

Evaluation criteria:

A test item is classified as mutagenic if it induced either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. However, the primary point of consideration is the biological relevance of the results. A test item that failed to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes was considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) were used as an aid in evaluating the results.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 4 animals (2 males, 2 females) received orally a single dose of 100 mg/kg b.w. test substance formulated in deionised water. The volume administered was 10 ml/kg b.w..
- Clinical signs of toxicity in test animals: reductions of spontaneous activity = 2/2 for all observation times; ruffled fur = 0/0 for all observation times except 2/2 at 6 h; Urine = orange at 2-4 h and 6 h.
- Rationale for exposure: In a GLP study (RCC-CCR study number 902402) performed parallel to this study the toxicity of the test item in Wistar rats after oral (gavage) application was analyzed. The data showed that one out of 4 animals died 24 h after treatment with 150 mg/kg b.w. Thus, the starting dose for the pre-experiment was set at 100 mg/kg b.w.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: See Table 1.
- Summary of Micronucleus Test: See Table 2
- Statistical Significance Data: Biometry: See Table 3

Any other information on results incl. tables

Table 1. Clinical signs of toxicity in test animals
Toxic Reactions		Hours post-treatment (male/female)
		1 hr	2-4 hr	6 h	24 h	48* h
100 mg/kg b.w. (vol administered is 10 ml/kg b.w.)	Reduction of spontaneous activity	7/7	10/10	12/12	12/12	1/1
	Ruffled fur	0/0	0/0	12/12	12/12	0/1
	Urine	–	orange	orange	dark yellow	–
50 mg/kg b.w. (vol administered is 10 ml/kg b.w.)	Reduction of spontaneous activity	0/0	3/3	3/4	0/0	N.O.
	Ruffled fur	0/0	0/0	4/4	3/3	N.O.
	Urine	–	orange	orange	dark yellow	N.O.
25 mg/kg b.w. (vol administered is 10 ml/kg b.w.)	Reduction of spontaneous activity	0/0	2/3	3/3	0/0	N.O.
	Ruffled fur	N.O.	N.O.	N.O.	N.O.	N.O.
	Urine	–	light orange	light orange	–	N.O.
* = only 6 animals per sex were used N.O. = not observed

Table 2. Summary of Micronucleus Test Results
Test Group	Dose (mg/kg b.w.)	Samling Time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythocytes
A. Stained with Giemsa/May-Grünwald
Vehicle	0	24	0.190	1-6	1088
Test Item	25	24	0.115	0-4	1079
Test Item	50	24	0.200	0-8	1081
Test Item	100	24	0.245	1-10	983
Positive Control	40	24	1.145	13-35	879
Vehicle	0	48	0.110	0-6	1035
Test Item	100	48	0.180	0-6	1053
B. Stained with Acridine
Vehicle	0	24	0.155	1-4	1134
Test Item	25	24	0.230	2-7	1237
Test Item	50	24	0.210	1-7	1107
Test Item	100	24	0.190	0-7	1076
Positive Control	40	24	1.990	15-66	784
Vehicle	0	48	0.150	0-5	1216
Test Item	100	48	0.145	1-6	1190

Table 3. Statistical Significance Data: Biometry
Vehicle Control vs. Test Group	Significance	p
A. Stained with Giemsa/May- Grünwald
25 mg test substance/kg b.w.; 24 h	n.t.	–
50 mg test substance/kg b.w.; 24 h	–	0.5
100 mg test substance/kg b.w.; 24 h	–	0.2147
40 mg CPA/kg b.w.; 24 h	+	< 0.0001
100 mg test substance/kg b.w.; 48 h	–	0.0627
B. Stained with Acridine
25 mg test substance/kg b.w.; 24 h	+	0.0286
50 mg test substance/kg b.w.; 24 h	–	0.0784
100 mg test substance/kg b.w.; 24 h	–	0.2719
40 mg CPA/kg b.w.; 24 h	+	< 0.0001
100 mg test substance/kg b.w.; 48 h	n.t.	–
Notes: Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test. – = not significant + = significant; n.t. = not tested, as the mean micronucleus frequency was not above the vehicle control value

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability). Under the conditions of the study, the test substance did not induce cytogenetic damage leading to micronucleus formation in the bone marrow of rats treated orally up to the maximal tolerated dose of 100 mg/kg, with clear demonstration that treated animals were exposed systemically. Therefore, the test substance was considered to be non-gentoxic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat. The test substance was formulated in degassed deionised water, which was also used as vehicle control. Two vehicle controls (one for 24 h and one for 48 h evaluation) were used. The volume administered orally was 10 mL/kg b.w. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

 

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

 

The following dose levels of the test item were investigated: 1) 24 h preparation interval: 25, 50, and 100 mg/kg b.w., 2) 48 h preparation interval: 100 mg/kg b.w. The highest dose (100 mg/kg) was estimated by a pre-experiment to be the maximum tolerated dose.

 

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance did not exert any cytotoxic effects in the bone marrow. However urine colour of the treated animals was orange to dark yellow indicating the systemic distribution of the test item, thus, confirming its bioavailbility. In addition, rats given 100 mg/kg ¹⁴C-test substance with the same regimen showed significant plasma levels 0.5 and 2 hours after compound administration (> 20 μg eg ¹⁴C-test substance/g of plasma).  In comparison to the corresponding vehicle controls there was no biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally as positive control showed a substantial increase of induced micronucleus frequency.