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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted similar to a guideline study and performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. Test material is commercial product not pure substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

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 *

Constituent 2

Reference substance name:

Ethylbenzene

EC Number:

202-849-4

EC Name:

Ethylbenzene

Cas Number:

100-41-4

Molecular formula:

C8H10

IUPAC Name:

ethylbenzene

Constituent 3

Reference substance name:

Xylene

EC Number:

215-535-7

EC Name:

Xylene

Cas Number:

1330-20-7

Molecular formula:

C8H10

IUPAC Name:

xylene isomers : Benzene, dimethyl- with ethylbenzene

Test material form:

liquid

Remarks:

Dark Red

Details on test material:

Purity:BAsed on name in report and physcial state and description, it is concluded that the commercial product (including solvent) was tested, rather than a solvent stripped dye.
Physical state at room temperature: Dark red liquid.
Stability: Unknown

Specific details on test material used for the study:

The test material appears to be the commercial product and thus contains a solvent (Ethylbenzene and xylene) at approximately 35% of the composition.

Test animals

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animal specification:
Swiss-Webster mice, 123 males and 123 females, born on May 16, 1989, were received by the SRI Laboratory Animal Medicine Department (LAMD) from Charles River Breeding Laboratories, Inc. (Colony POl) on June 14, 1989. The weights of 10 male and 10 female mice selected randomly ranged from 16.5 to 19.3 g and 14.2 to 16.1 g, respectively, at the time the animals were received. These animals were used for the range-finding study conducted the week of June 25, 1989. swiss-Webster mice, 120 males and 119 females, born on July 18, 1989, were received on August 16, 1989. The weights of 10 male and 10 female mice selected randomly ranged from 13.0 to 14.7 g and 12.0 to 13.2 g, respectively, at the time the animals were received. These animals were used for the definitive study conducted the week of August 27, 1989.

Test System Identification:
The animals were randomized and uniquely identified by ear punch. Cards attached to the outside of each cage contained the study number, test group and subgroup number, dose, and the ear-punch numbers of the animals housed in that cage.

Supplier:
Charles River Breeding Laboratories, Inc. [Colony POl] Shaver Road, Portage, MI 49081

Quarantine:
Mice received on June 14, 1989, and August 16, 1989, were quarantined for seven days and were released on June 21, 1989, and August 23, 1989, respectively. No sign or evidence of significant clinical disease was observed at any time during the quarantine periods or during the study. No notable gross pathologies were found in either 12 male or 12 female mice that were necropsied during the two quarantine periods. Mice not used for this particular study were assigned to concurrently conducted studies within the same project.

Animal Room Environmental Conditions:
Rooms: Building L, Rooms W110 and W107.
Temperature range: 60.8 to 84°F.
Humidity range: 38 to 81%.
Light cycle: 12 hours light/12 hours dark.
Cage specification: Mice were housed no more than 10/cage during quarantine and 5/cage during test period in polycarbonate cages containing hardwood-chip bedding.

Food and water Supply:
Food: Purina certified Rodent Chow #5002 ad libitum. Ralston Purina Co., st. Louis, MO. Lot Nos. APR5891C, MAR30891A, MAY23892B, MAY23892A.
Water: Purified tap water ad libitum via automatic watering system. Water-purity analysis on file, currently in Building 203/45.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): The total volume of test suspension administered per kilogram of body weight was 10 ml.

Negative Controls:
Name: Corn oil.
Lot no.: FEB1490L.
storage conditions: stored at 4°C.
Received: December 20, 1988.
Expiration: December 20, 1989.
Supplier: Mazola
Mission Supermarkets, Best Foods
CPC International, Inc.
Englewood Cliffs, NJ 07632.
Disposition: Retained as vehicle control.

Name: Corn oil.
Lot no.: L12590.
Storage conditions: Stored at 4°C.
Received: July 28, 1989.
Expiration: July 28, 1990.
Supplier: Springfield
PW Supermarkets
Certified Grocers of Calif., LTD.
Los Angeles, CA 90040
Disposition: Retained as vehicle control.

Details on exposure:

Test Article:
Name: Automate Red B.
Lot no.: 7111-2829.
MATERIALS
Purity: Sponsor assumes responsibility.
Physical state at room temperature: Dark red liquid.
Stability: Sponsor assumes responsibility.
Storage conditions: Stored in a secondary lightproof container at room temperature.
Received: June 15, 1989.
Disposition: Return to Sponsor when Final Report is issued.

Treatment:
The route of dosing, the method of administration, and the frequency of administration of the test articles were chosen (in consultation with the Sponsor) to maximize exposure of the animal to the test chemical. The test articles were prepared immediately prior to dosing.

A preliminary dose-range assay was performed to determine the appropriate dose levels for the definitive micronucleus study. Male and female swiss-Webster mice were given a single dose of the test article by oral intubation (gavage) on Days 1, 2, 3, and 4 and were sacrificed on Day 5. Mice, weighed individually, were dosed (three/group) with Automate Red B at 0, 300, 600, 1200, 2500, or 5000 mg/kg body weight (BW).

For the definitive assay, the test chemical was suspended in corn oil and administered by gavage at 0, 1200, 2500, 5000 mg/kg BW to 5 males and 5 females per dose group. The total volume of test suspension administered per kilogram of body weight was 10 mI. Mice were dosed with Automate Red B for four consecutive days and sacrificed approximately 24 hours after the final dose of Automate Red B. Five males also received the positive control Benzene at a dose of 500 mg/kg BW.

Duration of treatment / exposure:

For the definitive assay, the test chemical was suspended in corn oil and administered by gavage at 0, 1200, 2500, 5000 mg/kg BW to 5 males and 5 females per dose group. The total volume of test suspension administered per kilogram of body weight was 10 mI. Mice were dosed with Automate Red B for four consecutive days and sacrificed approximately 24 hours after the final dose of Automate Red B. Five males also received the positive control Benzene at a dose of 500 mg/kg BW.

Frequency of treatment:

Mice were dosed with Automate Red B for four consecutive days.

Post exposure period:

Mice were dosed with Automate Red B for four consecutive days and sacrificed approximately 24 hours after the final dose of Automate Red B.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Five/sex/dose group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive Control: Benzene (500 mg/kg in corn oil) was the positive control. The purpose of the positive control is to ensure proper conduct of the assay procedures. The positive control agent was administered only to 5 male mice

Name: Benzene (99+% pure, 500 mg/kg BW).
Lot no.: 03825EV.
Received: October 10, 1988.
Expiration: October 10, 1998.
Storage conditions: Stored at room temperature.
Supplier: Aldrich Chemical
940 W. st. Paul Avenue
Milwaukee, WI 53233
Disposition: Retained as positive control.

Examinations

Tissues and cell types examined:

Peripheral Blood Micronucleus Assay:
Blood samples were obtained by pricking the ventral tail vessel with a 25-gauge needle and drawing 2-3 ul of blood into a capillary tube. The sample was transferred to a clean microscope slide, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until staining. Three slides were prepared from each animal. Immediately prior to scoring, one of the three coded slides from the test animal was stained with acridine orange (Hayashi et al., 1983).

Details of tissue and slide preparation:

Data Collection:
Slides for micronucleus evaluation were coded using random letter codes generated by an IBM PC computer program. Slide labels were printed directly from the computer. Slides were coded by an individual not involved in the microscopic evaluation.

cytological Analysis:
Blood smears were evaluated Two parameters were determined: RNA-positive erythrocytes among using epifluorescence microscopy. (1) the number of micronucleated a total of 1000 RNA-positive erythrocytes per animal, which provides an index of chromosomal damage, and (2) the number of RNA-positive erythrocytes among 5000 erythrocytes per animal, which provides an index of cytotoxicity to the nucleated erythrocyte precursors. The criteria for micronuclei are those described by Schmid (1976), with the additional requirement that they exhibit the fluorescent characteristic of the staining combination (i.e., bright yellow in the case of acridine orange stain). The ratio of RNA-containing erythrocytes to mature erythrocytes (RBC) was based on the number of RNA-positive cells among approximately 5000 erythrocytes. Data from a given slide were entered directly into an IBM PC computer data file while scoring. After analyses were completed, the slides were decoded and the data were summarized using a decoding program on the IBM PC.

Evaluation criteria:

Criteria for a Valid Assay:
The data from this assay were considered acceptable if the frequency of micronucleated cells in the vehicle control group was within the normal historical range, if the positive control article resulted in a statistically significant elevation in the incidence of micronucleated cells, and if there were a minimum of three surviving animals of each sex with a percentage of RNA-positive erythrocytes greater than or equal to 15% of the control value.

Interpretation of Response:
Positive: The test article is considered unequivocally positive if the incidence of micronucleated RNA-containing erythrocytes is significantly higher than in the vehicle control group (p < 0.05) in two different dose groups. A positive dose-related increase in the incidence of micronucleated cells will also be considered a positive response.

Negative: The test article is considered unequivocally negative if the criteria for a positive or inconclusive response are not met.

Inconclusive: The results of this assay will be considered inconclusive if there is reason to believe that the concentrations of the test article selected for evaluation were inappropriate (e.g., excessive toxicity) or if a statistically significant elevation in micronucleated RNA-containing erythrocytes is observed in only one treatment group and the dose-response trend is not significant.

Statistics:

Statistical Tests Employed:
Data from each sex were analyzed both separately and combined, unless a statistically significant sex difference was observed between the vehicle control groups. The frequency of micronucleated RNA-containing erythrocytes among RNA-positive erythrocytes (i.e., the frequency of micronucleated PCES) and the percentage of RNApositive erythrocytes among total erythrocytes were calculated for each animal. The statistical significance of differences in the percentage of RNA-positive erythrocytes among groups was evaluated using the Kruskall-Wallace analysis of variance on ranks. The micronucleus frequency data were analyzed using the CochranArmitage test for trend in binomial proportions, to determine if a significant dose-response relationship was present, and the normal test for equality of proportions, to determine if individual dose groups were statistically elevated above controls. These tests and their rationale are discussed in the ASTM Standard Guide for Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes (ASTM Committee, 1988).

Results and discussion

Test results

Test results 1

Sex:

female

Genotoxicity:

negative

Remarks:

All dose levels.

Toxicity:

not specified

Remarks:

No toxicity observed in the preliminary study.

Vehicle controls validity:

valid

Negative controls validity:

other: Vehicle control was also negative control.

Positive controls validity:

valid

Test results 2

Sex:

male

Genotoxicity:

negative

Remarks:

1200 and 2500 mg/kg/ BW

Toxicity:

not specified

Remarks:

No toxicity observed in the preliminary study.

Vehicle controls validity:

valid

Negative controls validity:

other: Vehicle control was also negative control.

Positive controls validity:

valid

Test results 3

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

5000 mg/kg BW

Toxicity:

not specified

Remarks:

No toxicity observed in the preliminary study.

Vehicle controls validity:

valid

Negative controls validity:

other: Vehicle control was also negative control.

Positive controls validity:

valid

Additional information on results:

Preliminary Study Results:
A preliminary dose-range assay was performed to determine the appropriate dose levels for the definitive micronucleus study. Male and female swiss-Webster mice were given a single dose of the test article by oral intubation (gavage) on Days 1, 2, 3, and 4 and were sacrificed on Day 5. Mice, weighed individually, were dosed (three/group) with Automate Red B at 0, 300, 600, 1200, 2500, or 5000 mg/kg body weight (BW). No notable clinical signs were observed, and no animals died as a result of Automate Red B administration. Treatment with the above doses produced polychromatic erythrocyte to red blood cell (PCE/RBC) ratios of 1.30, 1.23, 1.39, 1.54, 1.42, and 0.91%, respectively, in males and 1.57, 1.27, 1.43, 1.29, 1.20, and 1.17%, respectively, in females. The high dose of Automate Red B was over 50% of the control PCE ratio (0.91% vs 1.30%, males; 1.17% vs 1.57%, females) and thus selected as the high dose for the definitive assay. No other statistical evaluations of the range-finding data were deemed necessary.

Definitive Study Results:
Doses of 0, 1200, 2500, and 5000 mg/kg BW of Automate Red B in males produced PCE/RBC values of 1.68, 1.80, 1.75, and 1.75%, respectively. In females, the same doses produced PCE/RBC values of 1.58, 1.75, 1.80, and 1.57%, respectively. There was no apparent decrease in the PCE/RBC ratios; therefore, no additional statistical methods were used to evaluate this parameter.
In male mice, doses of 1200, 2500, or 5000 mg/kg BW of Automate Red B yielded 0.16, 0.22, and 0.52% PCE with MN, respectively, compared with a vehicle control value of 0.20%. In females, the same doses yielded 0.12, 0.20, and 0.16% PCE with MN, respectively, compared with a vehicle control value of 0.26%. In contrast, benzene yielded 3.90% in male mice. The increase in micronucleus frequency in male mice was statistically significant at the high dose, but not at the mid and low doses. The Cochran-Armitage trend test indicated a positive trend for a dose-response relationship; however, this effect is clearly produced by the increase at the high dose.
Because a positive response was observed only at the high dose in male mice, slides from the control and high-dose groups for male mice treated in the range-finding assay (three per group) were rescored for micronuclei to confirm the positive response. This evaluation yielded 0.26 and 0.66% micronucleated PCE for doses of 0 and 5000 mg/kg BW, respectively, a statistically significant elevation.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive/Equivocal
The peripheral erythrocyte micronucleus (MN) assay was used to evaluate the clastogenic potential of Automate Red B. On the basis of the results, it is concluded that Automate Red B induces MN in polychromatic erythrocytes from swiss-Webster mice under the conditions of this assay. The response induced is relatively weak. Benzene produced roughly a 20-fold elevation in micronucleus frequency over controls at a dose of 500 mg/kg. In contrast, Automate Red B produced approximately a 2.5-fold elevation at 5000 mg/kg, making it approximately 80-fold less potent than benzene.

Executive summary:

SRI International assessed the ability of Automate Red B to induce micronuclei (MN) in peripheral erythrocytes following oral administration to male and female swiss-Webster mice. This study was conducted in compliance with the Good Laboratory Practice regulations proposed on December 28, 1987, by the United States Environmental Protection Agency Toxic Substances Control Act

(TSCA; 40 CFR 792). The laboratory work was begun on June 26, 1989, and completed on October 12, 1989. The study was initiated on June 19, 1989, and will be completed when the Final Report is issued.

A range-finding assay was performed to determine the doses used for this study. Mice were dosed, three per group, on Day 1 with Automate Red B suspended in corn oil at 0, 300, 600, 1200, 2500, or 5000 mg/kg body weight (BW) for four consecutive days. Surviving animals were sacrificed approximately 24 hours after the final dose. The number of RNA-positive erythrocytes (polychromatic erythrocytes, PCE) in a field of 5000 red blood cells (RBC) was determined. Only slight suppression of the PCE/RBC ratio was observed at 5000 mg/kg BW in males, and no suppression was observed in females at the same dose. Therefore, the doses selected for the definitive assay were 1200, 2500, and 5000 mg/kg BW. The positive control selected for the definitive assay was benzene (500 mgjkg).

Mice treated with the vehicle control in the definitive assay yielded a MN frequency of 0.20% in males and 0.26% in females. Doses of 1200, 2500, and 5000 mg/kg BW of Automate Red B yielded 0.16, 0.22, and 0.52%, respectively, in males and 0.12, 0.20, and 0.16%, respectively, in females. In contrast, the positive control, benzene, yielded 3.90% PCE with MN in male mice. The increase in MN frequency in male mice was statistically significant at the high dose, but not at the mid and low doses. Slides from the control and

high-dose groups for male mice treated in the range-finding assay evaluation yielded 0.26 and 0.66% micronucleated PCE for doses of 0 and 5000 mg/kg BW, respectively, a statistically significant elevation.

Based on the data obtained from the micronucleus assay, we conclude that Automate Red B induces an increase in micronuclei in peripheral erythrocytes.