2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 November 1987 to 29 January 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals (1981) No. 474 "Genetic Toxicology: Micronucleus Test"

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

EC Number:

284-395-7

EC Name:

2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

Cas Number:

84870-65-5

Molecular formula:

C19H18N6O2

IUPAC Name:

2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

Test material form:

solid

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

mouse

Strain:

other: albino CFLP

Details on species / strain selection:

Thirty-seven male and thirty-seven female albino CFLP strain mice were supplied by Interfauna (UK) Limited, Wyton, Huntingdon, Cambridgeshire.
The results of the test are believed to be of value in predicting the mutagenic potential of the test material to man. The test system was chosen because the mouse has been shown to be a suitable model for this type of study and is recommended in the test method .

Sex:

male/female

Details on test animals or test system and environmental conditions:

Thirty-seven male and thirty-seven female albino CFLP strain mice were supplied by Interfauna (UK) Limited, Wyton, Huntingdon, Cambridgeshire. At the start of the main study the males weighed 26 – 29 g, and the females 25 – 29 g, and were approximately five to eight weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a unique number within the study by ear punching and a number written on a colour coded cage card.
The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with sawdust bedding. With the exception of a 3-4 hour fast immediately before dosing and for approximately two hours after dosing, free access to mains drinking water and food (Rat and Mouse Expanded Diet No. 1, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.
The animal room was maintained at a temperature of 20 – 22 °C and relative humidity of 45 - 60%. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

For the purpose of this study the test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil B.P.

The vehicle was supplied by C.P. Pharmaceuticals Limited, as follows:
Description: clear, straw-coloured oily liquid
Container: plastic screw-top bottle
Supplier's identification: Oil Arachis B.P.
Batch number: 8611797
Storage conditions: room temperature
The identification and stability of the vehicle control were not determined.

Details on exposure:

For the purpose of this study the test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil B.P.

Duration of treatment / exposure:

Single treatment/ 24, 48 & 72 hours

Frequency of treatment:

One treatment

Post exposure period:

Animals were killed 24, 48 or 72 hours after dosing.

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

maximum practical dose

No. of animals per sex per dose:

30 animals (10 in each test group [5 male/5 female])
30 animals (10 in each vehicle control group [5 male/5 female])
10 animals (positive control [5 male/5 female])

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

The positive control material was supplied by Sigma Chemical Company, as follows:
Description: white powder
Container: brown glass screw-top bottle
Supplier's identification: Cyclophosphamide Monohydrate
Batch number: 85F-0054
Date of arrival: 26 June 1987
Storage conditions: + 4 °C in the dark
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water.
The identification and stability of the control material and the preparation was not determined.

Route of administration: Oral: gavage
Dose: 50 mg/kg bw
Concentration: 5 mg/mL
Dose volume: 10 mL/kg

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes were scored for the presence of micronuclei.

Details of tissue and slide preparation:

Immediately following sacrifice (i.e. 24, 48 or 72 hours following dosing), one femur was dissected from each animal, aspirated with foetal calf serum (Flow Laboratories Ltd) and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, defatted in xylene and stained in May-Gruenwald/Giemsa.
Evaluation of Slides
Stained bone marrow smears were examined at random using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 1000 polychromatic erythrocytes (blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (pink stained mature cells) associated with 1000 polychromatic erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of normochromatic to polychromatic erythrocytes was calculated together with appropriate group mean values for males and females separately and combined.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the number occurring in the corresponding vehicle control groups.
A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24, 48 or 72 hour kill times.
If the above criteria is not demonstrated, the test material is considered to be non-mutagenic under the conditions of the test.
A positive response for bone marrow toxicity is demonstrated when the treatment group mean normochromatic to polychromatic ratio is twice the vehicle control value or when a treatment related increase is shown to be statistically significant.

Statistics:

If necessary, and where possible, all data were statistically analysed using the Kruskal-Wallis one-way analysis of variance by ranks (Kruskal W.H. and Wallis W.A. 1952 J. Am. Statist. Soc. 47 583).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING TOXICITY STUDY
Mice treated with Dispersionsblau F-60 768 at 5000 mg/kg, showed no signs of toxicity and therefore 5000 mg/kg was selected as the “maximum practical dose”. No deaths or abnormal clinical observations were recorded.

MICRONUCLEUS STUDY
Mortality Data and Clinical Observations
No deaths abnormal clinical observations were recorded.

Evaluation of Bone Marrow Slides
There were no significant differences between the test material treatment groups and their corresponding vehicle control groups, with regard to the number of micronucleated polychromatic erythrocytes and micronucleated normochromatic erythrocytes.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the known mutagenic activity of cyclophosphamide monohydrate under the conditions of the test.
The test material did not induce an increase in micronucleated PCE’s that was consistent with a mutagenic effect. Dispersions-blau F-60 768 was, therefore, considered to be non-mutagenic under the conditions of the test in which no bone-marrow toxicity was observed.

Any other information on results incl. tables

RANGE-FINDING TOXICITY STUDY

The mortality data are summarised as follows:

DOSE LEVEL mg/kg	SEX	DEATHS ON DAY				TOTAL DEATHS
		0	1	2	3
5000	MALE	0	0	0	0	0/4
	FEMALE	0	0	0	0

 

MICRONUCLEUS STUDY

 

SUMMARY OF GROUP MEAN DATA (MALES AND FEMALES COMBINED)

TREATMENT GROUP	NUMBER OF PCE WITH MICRONUCLEI		NUMBER OF NCE WITH MICRONUCLEI^a		NCE RATIO PCE
	PER 1000 PCE		PER 1000 NCE
	GROUP MEAN	SD	GROUP MEAN	SD	GROUP MEAN	SD
1. DISPERSIONSBLAU 5000 mg/kg 24-hour sampling time	2.2	1.0	1.9	1.3	0.81	0.14
2. DISPERSIONSBALU 5000 mg/kg 49-hour sampling time	2.0	1.2	0.7	1.0	0.75	0.15
3. DISPERSIONBALU 5000 mg/kg 72-hour sampling time	2.5	1.9	1.9	1.3	0.57	0.13
4. VEHICLE CONTROL 24-hour sampling time	2.4	1.3	1.3	1.1	0.78	0.31
5. VEHICLE CONTROL 48-hour sampling time	1.9	1.2	0.9	0.7	0.93	0.22
6. VEHICLE CONTROL 72-hour sampling time	1.7	1.2	0.9	0.8	0.71	0.18
7. POSITIVE CONTROL 24-hour sampling time	44.9	6.2	3.3	0.9	1.39	0.38

Key:

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

SD = standard deviation

^a= calculated data

 

MICRONUCLEUS STUDY – INDIVIDUAL AND GROUP MEAN DATA

TREATMENT GROUP	ANIMAL NUMBER & SEX	TOTAL CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERYTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATIO
			NUMBER SCORED	PCE+MN	NUMBER SCORED	NCE+MN	NCE+MN PER 10^3NCE
DISPERSIONSBLAU 5000 mg/kg 24-hour sampling time	1 M 2 M 3 M 4 M 5 M	1699 1754 1745 1610 1857	1000 1000 1000 1000 1000	3.0 2.0 3.0 1.0 3.0	699 754 745 610 857	1.0 2.0 2.0 1.0 3.0	1.43 2.65 2.68 1.64 3.50	0.70 0.75 0.75 0.61 0.86
	MALE MEANS S.D.	1733 90	1000 0	2.4 0.9	733 90	1.8 0.8	2.38 0.85	0.73 0.09
	6 F 7 F 8 F 9 F 10 F	1888 1797 2119 1835 1752	1000 1000 1000 1000 1000	2.0 4.0 1.0 1.0 2.0	888 797 1119 836 752	0.0 3.0 0.0 2.0 1.0	0.00 3.76 0.00 2.40 1.33	0.89 0.80 1.12 0.84 0.75
	FEMALE MEANS S.D.	1878 144	1000 0	2.0 1.2	878 144	1.2 1.3	1.50 1.62	0.88 0.14
	GROUP MEANS S.D.	1806 136	1000 0	2.2 1.0	806 136	1.5 1.1	1.94 1.30	0.81 0.14

S.D. = standard deviation

TREATMENT GROUP	ANIMAL NUMBER & SEX	TOTAL CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERYTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATIO
			NUMBER SCORED	PCE+MN	NUMBER SCORED	NCE+MN	NCE+MN PER 10^3NCE
DISPERSIONSBLAU 5000 mg/kg 48-hour sampling time	11 M 12 M 13 M 14 M 15 M	1720 1772 1620 1841 1531	1000 1000 1000 1000 1000	0.0 2.0 4.0 2.0 3.0	720 772 620 841 531	1.0 2.0 0.0 0.0 0.0	1.39 2.59 0.00 0.00 0.00	0.72 0.77 0.62 0.84 0.53
	MALE MEANS S.D.	1697 123	1000 0	2.2 1.5	697 123	0.6 0.9	0.80 1.17	0.70 0.12
	16 F 17 F 18 F 19 F 20 F	1634 1897 2037 1644 1843	1000 1000 1000 1000 1000	2.0 2.0 1.0 3.0 1.0	634 897 1037 644 843	1.0 0.0 0.0 1.0 0.0	1.58 0.00 0.00 1.55 0.00	0.63 0.90 1.04 0.64 0.84
	FEMALE MEANS S.D.	1811 172	1000 0	1.8 0.8	811 172	0.4 0.5	0.63 0.86	0.81 0.17
	GROUP MEANS S.D.	1754 153	1000 0	2.0 1.2	754 153	0.5 0.7	0.71 0.97	0.75 0.15

S.D. = standard deviation

TREATMENT GROUP	ANIMAL NUMBER & SEX	TOTAL CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERYTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATIO
			NUMBER SCORED	PCE+MN	NUMBER SCORED	NCE+MN	NCE+MN PER 10^3NCE
DISPERSIONSBLAU 5000 mg/kg 72-hour sampling time	21 M 22 M 23 M 24 M 25 M	1529 1336 1595 1820 1614	1000 1000 1000 1000 1000	4.0 6.0 4.0 2.0 4.0	529 336 595 820 614	1.0 1.0 2.0 3.0 1.0	1.89 2.98 3.36 3.66 1.63	0.53 0.34 0.60 0.82 0.61
	MALE MEANS S.D.	1579 174	1000 0	4.0 1.4	579 174	1.6 0.9	2.70 0.90	0.58 0.17
	26 F 27 F 28 F 29 F 30 F	1672 1488 1432 1603 1602	1000 1000 1000 1000 1000	0.0 1.0 1.0 1.0 2.0	672 488 432 603 602	1.0 0.0 1.0 1.0 0.0	1.49 0.00 2.31 1.66 0.00	0.67 0.49 0.43 0.60 0.60
	FEMALE MEANS S.D.	1559 97	1000 0	1.0 0.7	559 97	0.6 0.5	1.09 1.04	0.56 0.10
	GROUP MEANS S.D.	1569 133	1000 0	2.5 1.9	569 133	1.1 0.9	1.90 1.25	0.57 0.13

S.D. = standard deviation

TREATMENT GROUP	ANIMAL NUMBER & SEX	TOTAL CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERYTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATIO
			NUMBER SCORED	PCE+MN	NUMBER SCORED	NCE+MN	NCE+MN PER 10^3NCE
VEHICLE CONTROL 24-hour sampling time	31 M 32 M 33 M 34 M 35 M	1459 1839 1600 1485 1593	1000 1000 1000 1000 1000	4.0 5.0 3.0 2.0 2.0	459 839 600 485 593	0.0 2.0 2.0 1.0 1.0	0.00 2.38 3.33 20.6 1.69	0.46 0.84 0.60 0.49 0.59
	MALE MEANS S.D.	1595 150	1000 0	3.2 1.3	594 150	1.2 0.8	1.89 1.22	0.60 0.15
	36 F 37 F 38 F 39 F 40 F	1907 1623 1703 2246 2381	1000 1000 1000 1000 1000	2.0 2.0 1.0 1.0 2.0	907 623 703 1246 1381	1.0 0.0 0.0 2.0 1.0	1.10 0.00 0.00 1.61 0.72	0.91 0.62 0.70 1.25 1.38
	FEMALE MEANS S.D.	1972 332	1000 0	1.6 0.5	972 332	0.8 0.8	0.69 0.70	0.97 0.33
	GROUP MEANS S.D.	1784 314	1000 0	2.4 1.3	784 314	1.0 0.8	1.29 1.13	0.78 0.31

S.D. = standard deviation

TREATMENT GROUP	ANIMAL NUMBER & SEX	TOTAL CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERYTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATIO
			NUMBER SCORED	PCE+MN	NUMBER SCORED	NCE+MN	NCE+MN PER 10^3NCE
VEHICLE CONTROL 48-hour sampling time	41 M 42 M 43 M 44 M 45 M	1963 1789 1915 1799 2053	1000 1000 1000 1000 1000	0.0 1.0 1.0 3.0 2.0	963 789 915 799 1053	0.0 0.0 1.0 1.0 1.0	0.00 0.00 1.09 1.25 0.95	0.96 0.79 0.92 0.80 1.05
	MALE MEANS S.D.	1904 112	1000 0	1.4 1.1	904 112	0.6 0.5	0.66 0.61	0.90 0.11
	46 F 47 F 48 F 49 F 50 F	1507 2019 2336 2050 1878	1000 1000 1000 1000 1000	4.0 1.0 3.0 2.0 2.0	507 1019 1336 1050 878	1.0 1.0 2.0 1.0 0.0	1.97 0.98 1.50 0.95 0.00	0.51 1.02 1.34 1.05 0.88
	FEMALE MEANS S.D.	1958 302	1000 0	2.4 1.1	958 302	1.0 0.7	1.08 0.74	0.96 0.30
	GROUP MEANS S.D.	1931 217	1000 0	1.9 1.2	931 217	0.8 0.6	0.87 0.67	0.93 0.22

S.D. = standard deviation

TREATMENT GROUP	ANIMAL NUMBER & SEX	TOTAL CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERYTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATIO
			NUMBER SCORED	PCE+MN	NUMBER SCORED	NCE+MN	NCE+MN PER 10^3NCE
VEHICLE CONTROL 72-hour sampling time	51 M 52 M 53 M 54 M 55 M	1598 1783 1608 1544 1861	1000 1000 1000 1000 1000	2.0 1.0 4.0 3.0 1.0	598 783 608 544 861	1.0 1.0 1.0 0.0 0.0	1.67 1.28 1.64 0.00 0.00	0.60 0.78 0.61 0.54 0.86
	MALE MEANS S.D.	1679 136	1000 0	2.2 1.3	679 136	0.6 0.5	0.92 0.85	0.68 0.14
	56 F 57 F 58 F 59 F 60 F	1548 1895 2047 1545 1631	1000 1000 1000 1000 1000	2.0 1.0 0.0 2.0 1.0	548 895 1047 545 631	0.0 1.0 0.0 1.0 1.0	0.00 1.12 0.00 1.83 1.58	0.55 0.90 1.05 0.55 0.63
	FEMALE MEANS S.D.	1733 226	1000 0	1.2 0.8	733 226	0.6 0.5	0.91 0.87	0.73 0.23
	GROUP MEANS S.D.	1706 178	1000 0	1.7 1.2	706 178	0.6 0.5	0.91 0.81	0.71 0.18

S.D. = standard deviation

TREATMENT GROUP	ANIMAL NUMBER & SEX	TOTAL CELLS SCORED (PCE+NCE)	POLYCHROMATIC ERYTHROCYTES (PCE)		NORMOCHROMATIC ERYTHROCYTES (NCE)			NCE PCE RATIO
			NUMBER SCORED	PCE+MN	NUMBER SCORED	NCE+MN	NCE+MN PER 10^3NCE
POSITIVE CONTROL 50 mg/kg 24-hour sampling time	61 M 62 M 63 M 64 M 65 M	2508 2366 1938 2817 2906	1000 1000 1000 1000 1000	43.0 44.0 42.0 41.0 54.0	1508 1366 938 1817 1906	5.0 4.0 4.0 2.0 7.0	3.32 2.93 4.26 1.10 3.67	1.51 1.37 0.94 1.82 1.91
	MALE MEANS S.D.	2507 387	1000 0	44.8 5.3	1507 387	4.4 1.8	3.06 1.20	1.51 0.39
	66 F 67 F 68 F 69 F 70 F	2404 2226 2840 1963 1905	1000 1000 1000 1000 1000	47.0 36.0 56.0 46.0 40.0	1404 1226 1840 963 905	6.0 4.0 6.0 3.0 3.0	4.27 3.26 3.26 3.12 3.31	1.40 1.23 1.84 0.96 0.91
	FEMALE MEANS S.D.	2268 378	1000 0	45.0 7.6	1268 378	4.4 1.5	3.45 0.47	1.27 0.38
	GROUP MEANS S.D.	2387 383	1000 0	44.9 6.2	1387 382	4.4 1.6	3.25 0.88	1.39 0.38

S.D. = standard deviation

Applicant's summary and conclusion

Conclusions:

The test material, Dispersionsblau F 60 768, was found not to produce micronuclei in polychromatic erythrocytes of mice under the conditions of the test.
The test substance is not classifiable according to CLP critera.

Executive summary:

A study was performed to assess the potential of Dispersionsblau F60 768 to produce damage to chromosomes or the mitotic apparatus of mice when administered by the oral route. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 474 “Genetic Toxicology: Micronucleus Test”.

 

Following a preliminary range-finding study to confirm the oral toxicity of the test material, the micronucleus study was conducted using Dispersionsblau F-60 768 at the maximum practical dose level (MPD).

 

In the micronucleus study, groups of ten mice (five males and five females) were given a single oral dose of Dispersionsblau F-60 768 at the maximum practical dose (5000 mg/kg). Animals were killed 24, 48 or 72 hours later. Polychromatic erythrocytes were scored for the presence of micronuclei.

 

Further groups of ten mice were treated with arachis oil B.P. or cyclophosphamide, to serve as vehicle and positive controls respectively.

 

There was no evidence of an increase in the incidence of micronucleated polychromatic erythrocytes in mice treated with Dispersionsblau F-60 768 when compared to the vehicle control groups. No statistical analysis was necessary.

 

The positive control material produced a very marked increase in the number of micronucleated polychromatic erythrocytes.

 

The test material, Dispersionsblau F-60 768, was considered to be non-mutagenic under the conditions of the test.

The test substance is not classified according to CLP criteria.