m-phenylenebis(methylamine)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-07-10 to 1989-08-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

feed was withheld overnight prior to dosing until approximately 8-9 hours after administration of the test substance, instead of 3-4 hours as stated in the protocol

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Deviations:

yes

Remarks:

see above

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

see above

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

m-phenylenebis(methylamine)

EC Number:

216-032-5

EC Name:

m-phenylenebis(methylamine)

Cas Number:

1477-55-0

Molecular formula:

C8H12N2

IUPAC Name:

1-[3-(aminomethyl)phenyl]methanamine

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Metaxylenediamine
- Physical state: Clear liquid
- Stability of test article: More than 6 months when under nitrogen atmosphere
- Stable for at least 4 hours in vehicles: Stable in water during 24 hours but precipitate in a carbon dioxide atmosphere. Therefore, the use of carbon dioxide during exposure time was avoided.
- Storage condition of test material: In the original container under nitrogen at room temperature in the dark
- Safety precautions: Gloves, goggles and face mask were considered sufficient to ensure personal health and safety

Test animals

Species:

mouse

Strain:

other: Swiss CD-1 (SPF quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Swiss mice, CD-1 (SPF quality)
- Source: Charles River Wiga GmbH, Sulzfeld, FRG
- Age at study initiation: Approximately 8 weeks
- Date of arrival of animals: 04 July 1989
- Identification: By unique cage number and a mark on the tail
- Assigned to test groups randomly: Yes, allocated to treatment groups as the animals came to hand from delivery boxes
- Weight at study initiation: 20-26 g (females), 26-34 g (males)
- Fasting period before study: Feed was withheld overnight prior to dosing until approximately 8-9 hours after administration of the test subtance.
- Housing: In groups of 5 per sex in polycarbonate cages.
- Bedding: Purified sawdust (Woody Clean, Broekmann Institute, Someren, The Netherlands)
- Diet: Standard Laboratory animal diet, RMH-B, pellet diameter 10 mm, Hope Farms, Woerden, The Netherlands. The feed was analysed for contaminants by the manufacturer.
- Water: Tap water, ad libitum. Results of chemical and contaminant analyses were archived.
- Acclimation period: At least 6 days under laboratory conditions


ENVIRONMENTAL CONDITIONS

- Temperature: 21 ± 3 degrees Centrigrade
- Humidity: 40-70 %
- Air changes: 7.5 per hour
- Photoperiod: 12 hours artificial fluorescent light and 12 hours darkness

Administration / exposure

Route of administration:

other: intubation

Vehicle:

- Milli-RO water

Details on exposure:

- Method: The test substance was administered by oral intubation.
- Rationale: The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and us
- Dosing volume: 10 mL/kg body weight

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Once

Post exposure period:

- Test animals were sacrificed at 24, 48 and 72 hours after dosing.
- Positive controls were sacrificed at 48 hours after dosing

Doses / concentrations

Remarks:

Doses / Concentrations:
750 mg/kg body weight
Basis:

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide (CP; CAS number 50-18-0 obtained from Endoxan, Astra-Werke, F.R.G) at 50 mg/kg body weight dissolved in 0.9 % sodium chloride (Merck) in Milli-RO water
- The route and frequency of administration and the volume administered were consistent with those of the test article.
- Solutions were prepared on the day of administration
- The stability of CP at room temperature is good. At 20 degrees Centigrade only 1 % of CP is hydrolysed per day in aqueous solution.

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

After the animals were sacrificed, both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 mL of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 minutes.

Evaluation criteria:

A test substance is considered positive in the micronucleus test if it induced a biologically as well as a statistically significant increase in the frequency of micronuclei (at any dose or sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucleus test if none of the testing concentrations or sampling times showed a statistically significant increase in the incidence of micronuclei neither in the combined data for both sexes nor in the data for male or female groups alone.

Statistics:

Wilcoxon Rank Sum Test; two sided test at p < 0.05

Results and discussion

Test results

Genotoxicity:

negative

Additional information on results:

Pilot study/dose selection

The animals of Group 1 did not show any signs of reaction to treatment. Animals of Group 2 and 3 recovered from the lethargy observed immediately post-dosing. In Group 4, three animals died within 48 hours post-dosing (one male, two females). In Group 5, all animals died within 24 hours post-dosing and in Group 6, all animals died within 4 hours post-dosing.

Based on the results of the pilot study, 750 mg/kg body weight was selected as an appropriate dose for the micronucleus test.

Main study

The mean bodyweights per group recorded immediately prior to dosing are presented in the Table 1 (attached).

The mean number of micronuclei per group and the mean ratio of polychromatic to normochromatics erythrocytes are presented in Table 2 (attached).

No increase in the frequency of micronuclei was observed.

Two male mice died between 2 and 3 days after dosing (one from Group E and one from Group F). The ratio of polychromatic/normochromatic erythrocytes does not evidence any toxic effect of the test substance on erythropoiesis.

The incidence of micronuclei in the control animals was found to be in the range of historical data (0.66 ± 0.93; mean ± standard deviation, N = 910).

The groups that were treated with with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a toxic effect of this compound on erythropoiesis.

Any other information on results incl. tables

Individual data are described in tabular form below. The mean number of micronuclei scored in the test substance-treated groups was compared with the corresponding control groups.

Micronucleus test: individual data

Group A-G males; oral dosing of metaxylenediamine

 

Group	Animal number	Number of micronuclei per 1000 polychromatic erythrocytes	Ratio polychromatic / normochromatic erythrocytes
A	2	0	0.86
A	4	0	0.93
A	6	0	1.00
A	8	1	0.86
A	10	0	0.89
B	12	0	1.07
B	14	0	0.77
B	16	0	1.02
B	18	0	1.02
B	20	1	0.92
C	22	0	1.15
C	24	0	0.92
C	26	1	1.05
C	28	0	0.98
C	30	1	1.01
D	72	0	0.88
D	74	0	0.96
D	76	0	0.90
D	78	1	1.08
D	80	0	0.87
E	82	2	1.01
E	84	0	0.84
E	86	1	0.95
E	88	2	0.98
E	90*	-	-
F	92*	-	-
F	94	0	0.95
F	96	0	0.90
F	98	0	0.88
F	100	0	0.89
G	62	12	0.30
G	64	6	0.27
G	66	8	0.25
G	68	9	0.36
G	70	10	0.34

 * Animal died after dosing.

Micronucleus test: individual data

Group A-G females; oral dosing of metaxylenediamine

 

Group	Animal number	Number of micronuclei per 1000 polychromatic erythrocytes	Ratio polychromatic / normochromatic erythrocytes
A	1	0	0.90
A	3	1	0.96
A	5	0	1.03
A	7	0	0.95
A	9	0	0.96
B	11	1	1.14
B	13	1	0.94
B	15	0	0.89
B	17	0	1.00
B	19	0	0.98
C	21	0	1.09
C	23	0	1.10
C	25	0	1.08
C	27	1	0.97
C	29	0	0.94
D	71	1	1.00
D	73	0	0.80
D	75	0	0.88
D	77	0	0.87
D	79	1	0.76
E	81	0	0.91
E	83	2	0.95
E	85	0	0.95
E	87	1	0.90
E	89	1	0.79
F	91	1	1.13
F	93	1	1.04
F	95	0	0.99
F	97	0	1.04
F	99	0	0.89
G	61	7	0.47
G	63	11	0.35
G	65	11	0.36
G	67	7	0.47
G	69	15	0.31

The positive control substance induced in both sexes a statistically significant increase in the number of micronuclei:

Wilcoxon rank-sum test

 

Number of micronuclei per 1000 polychromatic erythrocytes; treatment/control comparison

 

Group	Treatment	Dose mg/kg body weight	Sex	p-value (two-sided)	Decision at 95 % confidence level
G	CP	50	Males	0.01	Significant
G	CP	50	Females	0.01	Significant

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that the test is valid and that metaxylenediamine can be considered as not mutagenic in the micronucleus test under the experimental conditions decribed.