N-(dodecylphenyl)naphthalen-1-amine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2001 to 12 November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test",

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Method B12 of the EEC Commission Directive 92/69/EEC

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian germ cell cytogenetic assay

Test material

Test material information

1

Reference substance name:

-

EC Number:

437-450-6

EC Name:

-

Cas Number:

64654-05-3

Molecular formula:

Hill formula: C28 H37 N

IUPAC Name:

N-(dodecylphenyl)naphthalen-1-amine

Test material form:

liquid: viscous

Details on test material:

Sponsor's identification: APAN
Description: redbrown viscous liquid
Lot number: EL01010B01/KZ8911.5
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

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:

Sufficient albino Crl:CD-1 TM(ICR)BRs train mice were supplied by Charles River (UK) Limited, Margate, Kent. At the start of the main study the mice weighed 24 to 30g and were approximately five to eight weeks old. After a minimum acclimatisation period of seven days the animals were selected at random and given a number unique 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 seven in solid-floor polypropylene cages with woodflakes bedding. Free access to mains drinking water and food (Certified Rat and Mouse Diet 5LF2, PMI Nutrition International, Nottingham, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

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

Details on exposure:

Groups, each of seven mice, were dosed once only via the intraperitoneal route with the test material.

Duration of treatment / exposure:

Single treatment, 24 or 48 hours exposure.

Frequency of treatment:

All animals were dosed once only.

Post exposure period:

48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Groups, each of seven mice, were dosed with the test material.
Three further groups of mice were included in the study; two groups (seven mice) were dosed via the intraperitoneal route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide.

Control animals:

yes

Positive control(s):

Supplier's identification: Cyclophosphamide
Supplier's lot number: 108H0568
Safepharm serial number: R-2 1 89
Date received: 05 June 2001
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 (Parkfield Pharmaceuticals batch no. A1 147).

Examinations

Tissues and cell types examined:

In mitotic cells in which chromosome damage has been caused by the test material or its metabolites, fragments (centric or acentric) or whole chromosomes tend to lag behind in the anaphase stage of cell division. After telophase, a large proportion of the fragments are not included in the nuclei of the daughter cells and hence form a single or multiple micronuclei (Howell-Jolly bodies) in the cytoplasm of these cells. These micronuclei are seen in a wide variety of cell types, but erythrocytes are chosen since micronuclei are easily detected in these cells.

Details of tissue and slide preparation:

Slide Preparation
Immediately following termination (ie. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

Slide Evaluation
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part 111 (1989). The data was analysed following a √(x + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results

Key result

Sex:

male

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
In animals dosed with test material via the oral and intraperitoneal routes no premature deaths or clinical signs were observed.
The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main study. Adequate evidence of test material toxicity was not demonstrated using either route of administration. Therefore, the intraperitoneal route was selected for use in the main study in an attempt to maximise exposure of the animals to the test material. The maximum recommended (MRD) of the test material, 2000 mg/kg, was selected for use in the main study, with 500 and 1000 mg/kg as the lower dose levels.

Micronucleus Study
Mortality Data and Clinical Observations
There were no premature deaths or clinical signs seen in any of the dose groups.

Evaluation of Bone Marrow Slides
Statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 2000 mg/kg test material groups when compared to their concurrent vehicle control groups. This was taken to indicate that a cytotoxic response had been achieved in the target issue (bone marrow).
There were statistically significant increases in the frequency of micronucleated PCEs at all three of the 24-hour test material dose groups when compared to their concurrent vehicle control group. However, all three values were well within the normal historical range for vehicle controls, and were only significant because of the unusually low vehicle control value.
Therefore, the increases were considered to have no toxicological significance.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophospharnide under the conditions of the test.
The test material was found not to produce any toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Any other information on results incl. tables

Micronucleus Study – Summary of Group Mean Data

TREATMENT GROUP	NUMBER OF PCE WITH MICRONUCLEI PER 200 PCE		PCE/NCE RATIO
	GROUP MEAN	SD	GROUP MEAN	SD
1. Vehicle Control 48-hour sampling time	0.9	1.2	0.91	0.21
2. Vehicle Control 24-hour sampling time	0.1	0.4	1.09	0.46
3. Positive Control 24-hour sampling	58.6***	36.2	1.23	0.39
4. APAN 2000 mg/kg 48-hour sampling time	0.3	0.5	0.58**	0.16
5. APAN 2000 mg/kg 24-hour sampling time	1.7***	0.8	0.62*	0.23
6. APAN 1000 mg/kg 24-hour sampling time	1.6**	0.8	0.90	0.22
7. APAN 500 mg/kg 24-hour sampling time	2.3*	2.4	0.77	0.18

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standard deviation

* = P <0.05

** = P <0.01

*** = P <0.001

 

Micronucleus Study – Individual and Group Means and Standard Deviations: Vehicle Control (10 ml/kg) 48-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
			NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM
1. VEHICLE CONTROL 10 ml/kg 48-hour sampling time	1	28	2000	0	0.00	626	0	0.60
	2	27	2000	0	0.00	493	0	1.03
	3	27	2000	0	0.00	468	0	1.14
	4	28	2000	3	0.15	529	1	0.89
	5	29	2000	1	0.05	522	0	0.92
	6	29	2000	2	0.10	606	0	0.65
	7	29	2000	0	0.00	471	1	1.12
	Group Mean	28.1	2000	0.9	0.04	531	0.3	0.91
	SD	0.9	0	1.2	0.06	63	0.5	0.21

 

Micronucleus Study – Individual and Group Means and Standard Deviations: Vehicle Control (10 ml/kg) 24-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
			NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM
2. VEHICLE CONTROL 10 ml/kg 24-hour sampling time	8	27	2000	0	0.00	590	0	0.69
	9	25	2000	0	0.00	378	0	1.65
	10	29	2000	0	0.00	375	0	1.67
	11	29	2000	0	0.00	541	0	0.85
	12	27	2000	0	0.00	630	0	0.59
	13	29	2000	1	0.05	423	0	1.36
	14	29	2000	0	0.00	547	0	0.83
	Group Mean	27.9	2000	0.1	0.01	498	0.0	1.09
	SD	1.6	0	0.4	0.02	104	0.0	0.46

 

Micronucleus Study – Individual and Group Means and Standard Deviations: Cyclophosphamide (50 mg/kg) 24-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
			NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM
3. CYCLOPHOSPHAMIDE 50 mg/kg 24-hour sampling time	15	28	2000	22	1.10	413	0	1.42
	16	30	2000	32	1.60	622	1	0.61
	17	30	2000	49	2.45	442	0	1.26
	18	28	2000	80	4.00	376	0	1.66
	19	29	2000	110	5.50	459	1	1.18
	Group Mean	29.0	2000	58.6	2.92	462	0.4	1.23
	SD	1.0	0	36.2	1.81	95	0.5	0.39

 

Micronucleus Study – Individual and Group Means and Standard Deviations: Test Material (2000 mg/kg) 48-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
			NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM
4. APAN 2000 mg/kg 48-hour sampling time	20	29	2000	1	0.05	698	2	0.43
	21	29	2000	0	0.00	661	1	0.51
	22	26	2000	0	0.00	565	0	0.77
	23	27	2000	0	0.00	556	1	0.80
	24	24	2000	0	0.00	615	0	0.63
	25	27	2000	1	0.05	657	1	0.52
	26	29	2000	0	0.00	728	0	0.37
	Group Mean	27.3	2000	0.3	0.01	640	0.7	0.58
	SD	1.9	0	0.5	0.02	65	0.8	0.16

 

Micronucleus Study – Individual and Group Means and Standard Deviations: Test Material (2000 mg/kg) 24-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
			NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM
5. APAN 2000 mg/kg 24-hour sampling time	27	29	2000	2	0.10	590	1	0.69
	28	28	2000	1	0.05	514	1	0.95
	29	24	2000	3	0.15	714	0	0.40
	30	29	2000	2	0.10	743	0	0.35
	31	27	2000	2	0.10	550	2	0.82
	32	29	2000	1	0.05	583	0	0.72
	33	25	2000	1	0.05	699	1	0.43
	Group Mean	27.3	2000	1.7	0.09	628	0.7	0.62
	SD	2.1	0	0.8	0.04	90	0.8	0.23

 

Micronucleus Study – Individual and Group Means and Standard Deviations: Test Material (1000 mg/kg) 24-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
			NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM
6. APAN 1000 mg/kg 24-hour sampling time	34	28	2000	2	0.10	481	0	1.08
	35	29	2000	0	0.00	535	1	0.87
	36	27	2000	1	0.05	593	1	0.69
	37	30	2000	2	0.10	456	0	1.19
	38	26	2000	2	0.10	487	0	1.05
	39	27	2000	2	0.10	623	0	0.61
	40	30	2000	2	0.10	557	0	0.80
	Group Mean	28.1	2000	1.6	0.08	533	0.3	0.90
	SD	1.6	0	0.8	0.04	62	0.5	0.22

 

Micronucleus Study – Individual and Group Means and Standard Deviations: Test Material (500 mg/kg) 24-Hour Sampling Time

TREATMENT GROUP	ANIMAL NUMBER	BODYWEIGHT (g) WHEN DOSED	POLYCHROMATIC ERYTHROCYTES (PCE)			NORMOCHROMATIC ERYTHROCYTES (NCE)		PCE/NCE RATIO
			NUMBER SCORED	PCE + MN	%PCE + MN	NUMBER SCORED	NCE + NM
7. APAN 500 mg/kg 24-hour sampling time	41	28	2000	6	0.30	620	1	0.61
	42	28	2000	0	0.00	574	0	0.74
	43	27	2000	0	0.00	477	0	1.10
	44	26	2000	4	0.20	518	0	0.93
	45	28	2000	3	0.15	633	0	0.58
	46	30	2000	0	0.00	590	2	0.69
	47	27	2000	3	0.15	579	0	0.73
	Group Mean	27.7	2000	2.3	0.11	570	0.4	0.77
	SD	1.3	0	2.4	0.12	55	0.8	0.18

 

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-genotoxic under the conditions of the test.

Executive summary:

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to comply with the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The study design also complies with the revised OECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test", Method B12 of the EEC Commission Directive 92/69/EEC, the USA EPA, TSCA and FIFRA guidelines and the Japanese METIMHLW guidelines for testing of new chemical substances.

 

A range-finding study was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes, therefore the main study was performed using only male mice. The micronucleus study was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum recommended dose (2000 mg/kg) with 1000 and 500 mg/kg as the two lower dose levels.

Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

 

Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively.

Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

 

Statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 2000 mg/kg test material dose groups when compared to their concurrent control groups.

This was taken to indicate that a cytotoxic response had been achieved in the target tissue (bone marrow).

 

There were statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material at all three dose levels used when compared to the concurrent vehicle control group. However, all three values were well within the normal historical range for vehicle controls (Appendix I), and were only significant because of the unusually low vehicle control value. Therefore, the increases were considered to have no toxicological significance.

 

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

 

Conclusion. The test material was considered to be non-genotoxic under the conditions of the test.