2-Naphthalenol, 1-[[4-(phenylazo)phenyl]azo]-, ar-heptyl ar',ar''-Me derivs.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-Naphthalenol, 1-[[4-(phenylazo)phenyl]azo]-, ar-heptyl ar',ar''-Me derivs.

EC Number:

296-120-8

EC Name:

2-Naphthalenol, 1-[[4-(phenylazo)phenyl]azo]-, ar-heptyl ar',ar''-Me derivs.

Cas Number:

92257-31-3

Molecular formula:

Not applicable

IUPAC Name:

2-​Naphthalenol, 1-​[2-​[4-​(2-​phenyldiazenyl)​phenyl]​diazenyl]​-​, ar-​heptyl ar',​ar''-​Me derivatives *

Test material form:

other: Dark resinous solid

Details on test material:

TEST MATERIAL:
The test material, 2-Naphthalenol[(phenylazo)phenyl]azo heptyl derivatives (hereafter referred to as 2-NAPA), a dark resinous solid labelled Batch 7111-3055, and of stated purity >99%, was received on 13 September 1989 from the Study Sponsor. A sub-sample, totalling 25 g of compound, was removed and contained in dark, glass, screw-capped jar; this was stored in the dark at ambient temperature until required.
The compound was found to be insoluble in DMSO, but was soluble in acetone up to a maximum concentration of approximately 400 mg/m1. 2-NAPA was therefore freshly dissolved in acetone before addition to test cultures. The solution of maximum concentration was prepared initially; lower concentrations were prepared from this by serial dilution in acetone. All concentrations and dosages cited in this report refer to the 2-NAPA sample as received; no allowance has been made for purity or activity below 100%.
No determinations of homogeneity and concentration were performed on prepared solutions of test compound.
Solutions of cyclophosphamide (Endoxana, W.B. Pharmaceuticals) in sterile distilled water, and chlorambucil (Sigma Chemicals) in ethanol, were prepared immediately prior to use and served as positive controls. Cyclophosphamide is converted in the presence of an S-9 mix activating system to a highly reactive, clastogenic form. Chlorambucil is a direct-acting clastogenic agent.

Method

Target gene:

Chromosome Abberation

Species / strain

Species / strain / cell type:

lymphocytes: Human

Metabolic activation:

with and without

Metabolic activation system:

Rat liver-derived metabolic activating system (S-9 mix)

Test concentrations with justification for top dose:

The concentrations used in the main study, for all exposure regimes, were 8, 80 and 800 ug/ml (the maximum practicable concentration).

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: chlorambucil

Details on test system and experimental conditions:

Culture establishment:
Human peripheral blood was obtained by venepuncture from a healthy, non-smoking, male, human volunteer not currently taking any medication, and collected in heparinised vessels. Small inocula of whole blood (0.5 ml) were added to tubes containing 9.0 ml of culture medium and 0.5 ml phytohaemagglutinin to stimulate lymphocytes to divide. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping. After 48 hours of incubation, cultures were treated as described below.

Treatment of cultures:
Each 48-hour culture was centrifuged, the supernatant removed and the cell pellet resuspended in culture medium (9.5 ml, or 8.5 ml for cultures subsequently to contain S-9 mix). Freshly prepared S-9 mix (1.0 ml) was then added to the appropriate cultures and an aliquot (20 ul) of test solution, solvent or positive control solution was added to the relevant cultures. All cultures were incubated at 37°C in a shaking water bath, for three hours. After this initial exposure period, the cultures treated in the absence of S-9 mix were transferred to a 37°C incubator for the remainder of the exposure period. Cultures treated in the presence of S-9 mix and appropriate non-activated cultures were centrifuged and the cells washed twice with Hanks' Balanced Salt Solution (5m1) to remove the test compound and S-9 mix. The cells were then resuspended in culture medium (9.5 ml), and the cultures incubated at 37°C, under static conditions, for a further 21 hours.

Culture harvesting:
Three hours before termination, cell division was arrested by the addition of the spindle poison Colcemid to each culture (to a final concentration of 0.4 ug/ml). The tubes were capped and left to incubate for a further three hours. The cells were then harvested by low speed centrifugation (1400 r.p.m. for 5 minutes) and the pellets of cells thus collected were resuspended in hypotonic potassium chloride solution (0.56%) for ten minutes, centrifuged again and later fixed in freshly prepared methanol : glacial acetic acid fixative (3 : 1 v/v).

Slide preparation:
After two further changes of fixative, the tubes were centrifuged, the supernatant removed and the cell pellet resuspended in a few drops of fresh fixative. Single drops of the cell suspension were transferred to clean, moist, grease-free glass slides, and the slides were left to air-dry. Two or four slides (for the preliminary toxicity test or main cytogenetic test respectively) were made from each culture, stained for ten minutes in Giemsa stain (1 in 10 in Sorensen's buffer, pH 6.8), washed in buffer and left to air-dry. Permanent mounts were made using DPX mountant after clearing in xylene.

Preparation of culture media:
443 ml RPMI 1640 medium with HEPES, sodium bicarbonate, and L-glutamine
50 ml Foetal calf serum
5 ml Heparin (100i.u./m1)
2 ml Penicillin/streptomycin (5000 i.u./ml; 5000 ug/ml)

Preparation of a post-mitochondrial fraction and S-9 liver enzyme mix:
1. Post-mitochondrial fraction
Young male CD rats, c. 200 g bodyweight, were obtained from Charles River Breeding Laboratories (U.K.), Margate, Kent, England. Aroclor 1254 (500 mg/kg bodyweight in corn oil) was administered as a single intraperitoneal injection to induce microsomal enzyme activity. Four days after treatment, the animals were fasted overnight and then killed by cervical dislocation. The livers were removed, washed in cold 0.15M KC1, then homogenised with one volume of the same medium in a Potter-Elvehjem homogeniser. Homogenates were centrifuged at 9000 G for 10 minutes, and supernatants collected and stored at -180°C until required for preparation of the S-9 mix. Supernatant is used within six months of preparation.

2. S-9 mix
0.1M KH2PO4-Na2HPO4 buffer 7.4 ml
0.4M MgC12.6H20/1.65M KC1 aqueous solution 0.2 ml
0.1M NADP, sodium salt, in aqueous solution 0.4 ml
0.1M glucose-6-phosphate, sodium salt, in aqueous solution 0.5 ml
Supernatant from liver homogenate (protein content 102 mg/ml) 1.5 ml

Protein concentration in S-9 mix : 15.3 mg/ml

Evaluation criteria:

Main cytoqenetic test:
- chromosomal analysis and mitotic index. At least two slides from each culture were randomly assigned code numbers by a person not subsequently engaged in the study. Care was taken to ensure that all unique identifications remained concealed to eliminate bias. The slides were examined under a low power (x 10 objective) and those areas judged to be of sufficient technical quality to permit scoring were located and examined under high power (x 100, oil immersion objective). From 100 metaphases (with 46 centromeres) the following characters were recorded:
- chromosome number
- all chromosomes normal or some aberrant
- specific types and numbers of aberrations
Scoring followed the recommendations of the Ad Hoc committee of the Environmental Mutagen Society and the Institute for Medical Research (1972). Morphological observations were scored as follows:
Gap
Break
Fragment
Exchange
Multiple aberrations
Endoreduplication
Pulverised metaphases
Polyploidy

To examine the toxicity of the test compound to dividing lymphocytes, approximately 1000 cells were scored and the mitotic index calculated.
When all examinations had been completed, the coding was broken and the results collated. Frequencies of aberrant metaphases (cells showing one or more aberrations) were calculated for each culture scored, both including and excluding gap-type aberrations.

Statistics:

Statistical analysis:
The Fisher Exact Probability test is a useful nonparametric technique for analysing data when comparing two small independent samples. It is used when the scores for the samples all fall into one or other of two mutually exclusive classes. The test determines whether the two groups differ in the proportions with which they fall into the two classifications. Data from each treatment was compared with the respective solvent control group with or without activation.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

After 48 hours, both cultures containing 2-NAPA at 800 ug/ml and one containing 80 ug/ml produced a number of metaphases which could not be clearly visualised under the light microscope. This event is believed to be indirectly associated with cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
Precipitation of test compound was apparent in cultures treated with 2-NAPA at 800 ug/ml. When slides were prepared, precipitated compound was apparent in the cell pellet of all cultures treated at 32, 160 and 800 ug/ml. Slides were prepared and stained from all cultures. Inspection of the slide preparations showed no real reduction in mitotic activity (compared to solvent control values) at any 2-NAPA concentration tested, except for a single culture treated at 160 ug/ml in the presence of S-9 mix, which was seen to contain cells but no metaphases. As no such effect was apparent in cultures treated at 800 ug/ml, this was not considered to be a treatment-related effect. Following the findings of the preliminary toxicity test, the 2-NAPA concentrations selected for testing in the main cytogenetic study were 8, 80 and 800 ug/ml for all exposure periods.

Main cytogenetic test:
Precipitation of test compound was apparent in all cultures treated at 800 ug/ml; the supernatant of all cultures treated at 80 ug/ml for 3 hours showed intense red colouration at the end of the exposure period.

Evidence of toxicity:
Initially, mitotic indices were scored for all cultures. No clear depression of mitotic index in test cultures was apparent in either the presence or absence of S-9 mix following any exposure period. However, after 48 hours of exposure, both cultures containing 2-NAPA at 800 ug/ml and one containing 80 ug/ml produced a number of metaphases which could not be clearly visualised under the light microscope (the chromosomes were 'fuzzy'). This event has been seen in other studies performed in this laboratory with other (unrelated) test materials at toxic concentrations, and it is believed to be indirectly associated with cytotoxicity.

Incidence of chromosomal aberrations: comparison of treated and control cultures
One hundred metaphases were scored from each culture. In the absence of S-9 mix, cultures exposed to the solvent (acetone) for three hours only showed incidences of aberrant metaphases of 5 and 6% with a mean of 5.5%. Corresponding values for cultures treated with 2-NAPA were 3 and 4%, mean 3.5% (at 8 ug/ml), 6% in both cultures tested at 80 ug/ml and 5 and 8%, mean 6.5% (at 800 ug/ml). In the presence of S-9 mix, cultures exposed to acetone for three hours only showed incidences of aberrant metaphases of 5 and 6% with a mean of 5.5%. Corresponding values for cultures treated with 2-NAPA were 4% in both cultures treated at 8 ug/ml, 5 and 13%, mean 9.0% (at 80 ug/ml) and 4 and 8%, mean 6.0% (at 800 ug/ml).
Following 24 hours of exposure in the absence of S-9 mix, solvent control cultures showed incidences of aberrant metaphases of 7 and 11% with a mean of 9.0%. Corresponding values for cultures treated with 2-NAPA were 6 and 9%, mean 7.5% (at both 8 and 80 ug/ml) and 9 and 10%, mean 9.5% (at
800 ug/ml). A similar lack of response to 2-NAPA treatment, at the above exposure periods, was apparent when gaps were excluded from consideration. These observations are supported by statistical analysis; no statistically significant increases in aberrant cell frequencies over control values were seen in cultures exposed to 2-NAPA at any concentration for either 3 or 24 hours whether including or excluding gaps (p>0.05 in all cases).
Following 48 hours of exposure in the absence of S-9 mix, solvent control cultures showed incidences of aberrant metaphases of 7 and 8% with a mean of 7.5%. Corresponding values for cultures treated with 2-NAPA were 5 and 7%, mean 6.0% (at 8 ug/ml), 9 and 12%, mean 10.5% (at 80 ug/ml) and
17 and 19%, mean 18.0% (at 800 ug/ml). When gap-type aberrations were excluded, solvent control cultures showed incidences of aberrant metaphases of 0 and 2% with a mean of 1.0%. Corresponding values for cultures treated with 2-NAPA were 0 and 1%, mean 0.5% (at 8 ug/ml), 1% in both cultures treated at 80 ug/ml and 2 and 4%, mean 3.0% (at 800 ug/ml).
No statistically significant increases were seen at any test concentration including gaps, or at 8 and 80 ug/ml excluding gaps (p>0.05). The observed increase in aberrant cell frequency, including gaps, in cultures exposed at 800 ug/ml was seen to be statistically significant (0.01>p>0.001).
When compared to the historical control range for this laboratory (shown below), solvent control cultures harvested after 24 and 48 hours of exposure to acetone showed a small excess of gap-type aberrations. The reason for this is unclear, but in view of the limited magnitude of this excess, the questionable biological significance of gaps and the low frequency of other types of aberration in all solvent control cultures (within the historical control range), this is not considered to impair the validity of the study in any way.

Solvent (acetone) controls: aberrant cell frequencies:
Historical values (600 cells): +S-9 mix 1-6% including gaps, 0-4% excluding gaps
Historical values (600 cells): -S-9 mix 2-6% including gaps, 0-3% excluding gaps

Positive controls:
Treatment with the known clastogen, cyclophosphamide, in the presence of S-9 mix, produced a marked increase in aberrant cell frequency, giving values of aberrant metaphases of 38 and 46%, with a mean of 42.0% including gaps, and values of 18 and 32%, mean 25.0% excluding gaps. Statistical analysis confirmed that this group had significantly more aberrant metaphases than the activated solvent controls whether gap-type aberrations were excluded or not (p < 0.001). Without S-9 mix, cyclophosphamide produced no increases in aberration frequencies compared with non-activated solvent controls whether gaps were included or not (p > 0.05).
Treatment with the known clastogen, chlorambucil, in the absence of S-9 mix showed increased aberrant cell frequencies following all exposure periods as shown below:

Exposure period Including gaps
(hours) Indiv. values p-value
3 16, 23 p<0.001
24 26, 29 p<0.001
48 16, 24 p<0.001

Exposure period Including gaps
(hours) Indiv. values p-value
3 5, 8 0.01>p>0.001
24 13, 14 p<0.001
48 5, 8 0.01>p>0.001

Polyploidy:
The incidence of polyploid cells throughout the study was low, and no treated group showed a real increase compared to concurrent solvent control cultures.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation All dose levels tested.
negative without metabolic activation All dose levels tested.

It is concluded that exposure to 2-NAPA at the highest tested concentration induced gap-type aberrations in human lymphocytes. This action was observed only in the absence of S-9 mix after prolonged (48 hours) exposure. In view of the questionable biological significance of gap-type aberrations, 2-NAPA would not be designated a clastogen on the basis of this evidence.

Executive summary:

The effects on chromosomal structure of exposure to 2-Naphthalenol [(phenylazo)phenyl]azo heptyl derivatives (2-NAPA) were investigated in cultured human lymphocytes. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activating system (S-9 mix): without S-9 mix cells were exposed for 3, 24 and 48 hours, with S-9 mix exposure was limited to three hours. Treatments were established by the addition of test solutions (in acetone) to 48 hour cultures established from whole, human blood. Cell division was arrested by the addition of the spindle poison, Colcemid (to a final concentration of 0.4 ug/ml), three hours before the cells were harvested; slides were then prepared for microscopic analysis.

Mitotic indices were calculated for each culture: these were based on the number of metaphases observed per 1000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture, and the frequencies of cells with one or more aberrations were calculated; these aberrant cell frequencies were calculated both including and excluding gap-type aberrations.

Following a preliminary test to assess toxicity of 2-NAPA to dividing lymphocytes,the concentrations used in the main study, for all exposure regimes, were 8, 80 and 800 ug/ml (the maximum practicable concentration).

The main test also incorporated solvent (acetone), and positive (cyclophosphamide and chlorambucil) control cultures. Cyclophosphamide is a known clastogen requiring biotransformation to achieve optimum activity; chlorambucil is a direct-acting clastogen. All control/test concentrations were established in duplicate cultures.

Consideration of mitotic index data showed no direct evidence of toxicity of 2-NAPA to dividing lymphocytes at any tested concentration in either the absence or presence of S-9 mix. However, after 48 hours of exposure, both cultures containing 2-NAPA at 800 ug/ml and one containing 80 ug/ml produced a number of metaphases which could not be clearly visualised under the light microscope (the chromosomes were 'fuzzy'). This event has been seen in other studies performed in this laboratory with other (unrelated) test materials at toxic concentrations, and it is believed to be indirectly associated with cyotoxicity.

No biological or statistically significant increases in the frequency of aberrant cells, over solvent controls, were recorded at any concentration following 3 or 24 hours of exposure (p>0.05); this was true whether gap-type aberrations were included in or excluded from analysis.

Following 48 hours of exposure, an increase in aberrant cell frequency was apparent in cultures treated with 2-NAPA at 800 ug/ml, but only when gaps were included; statistical analysis showed this increase to be significant (0.01>p>0.001)

The known clastogens, cyclophosphamide and chlorambucil, induced significant increases in chromosomal damage over concurrent vehicle controls for all exposure regimes; as expected, cyclophosphamide was active only in the presence of S-9 mix.

It is concluded that exposure to 2-NAPA at the highest tested concentration induced gap-type aberrations in human lymphocytes. This action was observed only in the absence of S-9 mix after prolonged (48 hours) exposure. In view of the questionable

biological significance of gap-type aberrations, 2-NAPA would not be designated a clastogen on the basis of this evidence.