m-phenylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro (Mutagenic effects - bacterial): NOAEL; OECD 471; Ames study; mutagenic; reliability = 2.
NOT CLASSIFIED

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

No reference to OECD guideline. Deviation from OECD guideline with the use of 4-o-Tolylazo-o-toluidine as positive control. Study conducted in 1975.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 strains of bacteria were used. Strain TA1538 was used and Strain TA98 was not. Positive controls deviate from those recommended in guideline.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitol-stimulated rat liver homogenate

Test concentrations with justification for top dose:

1, 10, 50, 100, 250, 500, 750, 1000 micrograms/plate.

Vehicle / solvent:

no data

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-o-Tolylazo-o-toluidine (for TA1538 and metabolic activation) and N-methyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

In the absence of metabolic activation, approximately 5E8 bacteria were added to 2 mL of top agar (0.6% Difco agar, 0.6% NaCl, 0.050 mM of L-histidine, 0.05 mM of biotin). After the test chemical was added to the top agar, the solution was mixed and poured onto the surface of a minimal agar plate with Vogel-Bonner E medium containing 1.5% agar and 2% glucose.

The metabolic activation system consisted of 0.15 mL of the 9000 x g supernatant of homogenized rat liver, MgCl2, KCl, glucose-6-phosphate, TPN, and a sodium phosphate buffer (pH 7.4). This mixture (0.25 mL) was added directly to the top agar immediately before it was poured over the minimal agar plate. The plates were incubated at 37 deg C for 40 hours. The number of histidine-positive (his+) revertant colonies on each plate then was counted. All tests were performed in duplicate.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION: 40 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF CELLS EVALUATED: 5E8

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

In Vitro Salmonella Typhimurium Assay With m-PDA.

 

Chemical	Metabolic Activation	Amount of Compound Added per Plate (µg)	Average Number of Histidine-Positive Revertants per Plate
			TA100	TA1535	TA1537	TA1538
Negative Control	-		72	11	5	9
	+		84	11	6	15
4-o-Tolylazo-o-toluidine (Positive control for TA1538 and metabolic activation)	-	25				10
	+	25				195
N-methyl-N’-nitro-N-nitrosoguanidine	-	2		600
m-phenylenediamine	-	1	65	9	4	4
	-	10	64	8	4	2
	-	50	78	9	5	9
	-	100	80	10	3	12
	-	250				9
	-	500	83	8	2	17
	-	750
	-	1000	74	11	5	21
	+	1	87	14	8	10
	+	10	78	11	7	17
	+	50	80	9	6	79
	+	100	85	14	6	177
	+	250				423
	+	500	84	14	11	570
	+	750				623
	+	1000	79	10	8	971

 

Conclusions:

The test substance was mutagenic in strain TA 1538 with metabolic activation in the S. typhimurium assay, but negative when activation was not used. It was negative in all other strains, with or without activation.

Executive summary:

The test substance was mutagenic in strain TA 1538 with metabolic activation in the S. typhimurium assay, but negative when activation was not used. It was negative in all other strains, with or without activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In Vivo (Clastogenic effects - mammalian): NOAEL; OECD 474; GLP; In vivo mouse micronucleus study; no mammalian mutagenesis was observed at any exposure level; reliability = 2.
NOT CLASSIFIED

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 6, 1990 to January 9, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. Conclusion based on scoring 1000 PCEs/animal for MNPCEs versus 2000 PCEs/animal as per OECD 474.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

scored fewer PCEs/animal than recommended.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Crl:CD-1(ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc., Breeding Area 62, Montreal, Quebec, Canada.
- Age at study initiation: 50 days old
- Weight at study initiation: Males were 28.6 to 33.5 g, females were 21.3 to 26.1 g
- Assigned to test groups randomly: By computer-generated random numbers
- Housing: Individually housed in standard wire mesh cages
- Diet: Purina certified Rodent Chow #5002, Lots # Aug 07 901A and Aug 10 901B) ad libitum
- Water: Ad libitum
- Acclimation period: Quarantined and acclimated for 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 50 ± 10
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

Deionized water

Frequency of treatment:

Twice, with doses administered approximately 24 hours apart.

Remarks:

Doses / Concentrations:
16, 33, 65 mg/kg/day
Basis: actual ingested

No. of animals per sex per dose:

5 males/females per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Details of tissue and slide preparation:

Immediately following sacrifice at 24 or 48 hours after the administration of the second dose, the marrow from both femurs of each animals was collected. A Miniprep automatic blood smearing instrument was used to make the bone marrow smears. At least 2 slides/animal were prepared.

Evaluation criteria:

Representative slides from each animal were examined in a blind manner using incident light fluorescence microscopy. Only cells showing good morphology and staining were selected for scoring. Polychromatic erythrocytes (PCEs) were identified by their characteristic reddish staining. Normochromatic erythrocyte (NCEs) appeared dark green. One thousand PCEs/animal were scored for the presence of micronuclei, which are typically round, bright yellow-green fluorescent bodies. Cellular inclusions that were irregularly shaped or stained, or were not in the focal plane of the cell were considered artifacts. The unit of scoring was the micronucleated cell. PCEs containing more than one micronucleus were counted as a single micronucleated cell. The number of micronucleated NCEs seen in the optic fields scored to obtain 1000 PCEs was also recorded. The proportion of PCEs among 1000 erythrocytes was determined for each animal.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 25, 50, 65, 75 mg/kg/day, 2X ~24 hours apart.

- Clinical signs of toxicity in test animals: 75 mg/kg/day group, 3/3 males and 1/3 females died within 30 hours of second dosing.
65 mg/kg/day-lethargic and had labored breathing; these signs were observed in all animals and occurred within 2 hrs of dosing on both days. Mild clinical signs (ruffled fur and partially closed eyes) persisted for 2 days following the final exposure, however, all animals recovered within 4 days post-treatment. 50 and 25 mg/kg/day-face pawing, labored breathing, gasping, lethargy and half closed eyes.

- Dose range: 85, 100, 200 or 300 mg/kg-a second treatment at these levels was not conducted due to excessive mortality or the severity of the clinical signs observed 24 hrs after the initial treatment


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: 65 mg/kg/day-within 1 hour of initial dose, mice exhibited lethargy, gasping, face pawing, spasms, tremors, or half-closed eyes. Lethargy, half-closed eyes and labored and/or rapid breathing were still evident in the majority of the animals, 3-4 hrs after initial observations. Clinical signs following the second dose were similar to those observed the preceding day. One male was found dead 24 hrs after the final treatment. 16 and 33 mg/kg/day-lethargy, half closed eyes and rapid breathing within 24 hrs of final dose. The majority of the 16 mg/kg/day treated group appeared to recover, and only slight clinical sign were still evident in the 33 mg/kg/day treated group.

Weight loss-there was a statistically significant weight loss in 33 and 65 mg/kg/day treated groups at the 24 hour sacrifice, and in the 65 mg/kg/day treated group at the 48 hr sacrifice.

There were no statistically significant increases in MN-PCEs among test substance treated mice at any dose level or sampling interval.

Table 1. Mouse Micronucleus Assay of m-PDA

 

m-PDA Treatment (mg/kg/day)	Sampling Time (hrs)	Sex	Animals Per Group	Change in Body Weight (g) x ± S.E.
0	24	M	5	0.0 ± 0.2
		F	5	-0.4 ± 0.5
16	24	M	4^a	-0.9 ± 0.6
		F	5	-0.7 ± 0.4
33	24	M	5	-3.3 ± 0.7**
		F	5	-2.8 ± 0.2**
65	24	M	6	-5.3 ± 0.4**
		F	6	-4.5 ± 0.3**
0	48	M	5	0.4 ± 0.4
		F	5	0.8 ± 0.9
65	24	M	4^a,b	-3.4 ± 0.9**
		F	6	-3.3 ± 0.5**
CP, 20	24	M	5	0.2 ± 0.3
		F	5	-0.6 ± 0.2

 

CP = cyclophosphamide

a animal misdosed and removed from study.

b animal died before scheduled sacrifice time.

** p < 0.01

 

 

Table 2. Data Summary. Mouse Micronucleus Assay of m-PDA.

 

m-PDA Treatement (mg/kg/day)	Sampling Time (hrs)	Mean % Micronucleated PCEs ± S.E.^a					Mean PCE:NCE Ratio ± S.E.
		N	Males	N	Females		Males		Females
0	24	5	0.16 ± 0.05	5	0.02 ± 0.02		1.16 ± 0.11		1.12 ± 0.03
16	24	4^b	0.15 ± 0.09	5	0.16 ± 0.05		0.96 ± 0.05		1.13 ± 0.08
33	24	5	0.24 ± 0.07	5	0.10 ± 0.04		0.91 ± 0.07		1.14 ± 0.07
65	24	6	0.28 ± 0.07	6	0.13 ± 0.05		0.77 ± 0.10		1.13 ± 0.12
0	48	5	0.40 ± 0.13	5	0.28 ± 0.05		0.98 ± 0.12		1.34 ± 0.09
65	48	4^b,c	0.50 ± 0.27	6	0.18 ± 0.03		0.58 ± 0.08*		0.87 ± 0.26
CP, 20	24	5	0.96 ± 0.09**	5	0.82 ± 0.09**		0.71 ± 0.03**		1.00 ± 0.10

 

CP = cyclophosphamide

a 1000 PCEs scored per animal.

b animal misdosed and removed from study.

c animal died before scheduled sacrifice time.

*   p < 0.05

** p < 0.01

 

Conclusions:

Mice exposed to the test substance did not exhibit statistically significant increases in micronucleated polychromatic erythrocytes at any dose level or sampling interval.

Executive summary:

No statistically significant increases in the frequency of micronucleated PCEs were observed in test substance-treated animals at any sampling time. A significant depression in the ratio of young, polychromatic erythrocytes to mature, normochromatic erythrocytes was observed in the high dose males at the 48 -hr sampling interval. Under the conditions of this assay, the test substance did not induce micronuclei; the test substance is negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test substance produced mutagenicity when evaluated in bacterial cultures, however it did not produce mutagenicity when evaluated in vivo (in animal studies). Further, the test substance was not carcinogenic in lifetime mammalian testing. 

Justification for classification or non-classification

The test substance produced mutagenicity when evaluated in bacterial cultures, however it did not produce mutagenicity when evaluated in vivo (in animal studies including an in vivo micronucleus test). Further, the test substance was not carcinogenic in lifetime mammalian testing. The substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. Although this classification deviates from the harmonized classification, the harmonized classification will be adhered as it is stricter.