Reaction mass of 2,4-bis(xylylazo)resorcinol and 2,4-bis[(4-dodecylphenyl)azo]resorcinol and 2-[(4-dodecylphenyl)azo]-4-(2,4-xylylazo)resorcinol

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in general conformance with applicable Environmental Protection Agency, Toxic Substances Control Act, Good Laboratory Practice standards with the following exceptions: 1. Test substance characterization and stability data were not developed by SRI International. 2. Assays to verify concentration, stability, and homogeneity of the test substance in the carrier vehicle were not performed. These deviations should not affect the results or conclusions of this study.

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:

Reaction mass of 2,4-bis(xylylazo)resorcinol and 2,4-bis[(4-dodecylphenyl)azo]resorcinol and 2-[(4-dodecylphenyl)azo]-4-(2,4-xylylazo)resorcinol

EC Number:

915-586-1

Molecular formula:

Variable; substance is a UVCB.

IUPAC Name:

Reaction mass of 2,4-bis(xylylazo)resorcinol and 2,4-bis[(4-dodecylphenyl)azo]resorcinol and 2-[(4-dodecylphenyl)azo]-4-(2,4-xylylazo)resorcinol

Test material form:

other: Liquid

Details on test material:

Name: Automate Yellow 8 Petroleum
Lot no.: 7146-266
Purity: Not included in report
Physical state at room temperature: Dark red liquid
Stability: Sponsor's responsibility
Storage conditions: Stored in a secondary lightproof container at room temperature

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 POI) 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.

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

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.

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 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
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:

Preliminary Dose-range Assay:
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 per group) with Automate Yellow 8 Petroleum at 0, 300, 600, 1200, 2500, or 5000 mg/kg body weight (BW).

Definitive Assay:
The test chemical was suspended in corn oil and administered by gavage at 0, 1200, 2500, or 5000 mg/kg BW. The total volume of test suspension administered per kilogram of body weight was 10 mI. Mice were dosed with Automate Yellow 8 Petroleum for four consecutive days and sacrificed approximately 24 hours after the final dose of Automate Yellow 8 Petroleum.

Duration of treatment / exposure:

Mice were dosed with Automate Yellow 8 Petroleum for four consecutive days and sacrificed approximately 24 hours after the final dose of Automate Yellow 8 Petroleum.

Frequency of treatment:

Mice were given a single dose of the test article by oral intubation (gavage) for four consecutive days and sacrificed approximately 24 hours after the final dose.

Post exposure period:

The mice were sacrificed approximately 24 hours after the final dose.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1200, 2500, or 5000 mg/kg BW
Basis:
nominal conc.

No. of animals per sex per dose:

5 animals/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

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 male mice.

Name: Benzene (99+% pure, 500 mgjkg 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:

The target cell population tested consists of erythroblasts undergoing their final chromosome replication and mitosis prior to expulsion of the main nucleus.

Cytological Analysis:
Blood smears were evaluated using epifluorescence microscopy. Two parameters were determined: (1) the number of micronucleated RNA-positive erythrocytes among 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 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.

Details of tissue and slide preparation:

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).

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.

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.

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 RNA-positive 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. If a significant overall difference was observed, the individual dose groups that differed from the control were determined by using a distribution-free multiple comparison test (Gad and Weil, 1986).
The micronucleus frequency data were analyzed as follows: In experiments in which frequencies of micronucleated cells are based on scoring 1000 cells per animal, data are not expected to be distributed normally. Such data are 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

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

All dose levels.

Toxicity:

no effects

Remarks:

All dose levels.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Dose Range Assay 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 per group) with Automate Yellow 8 Petroleum at 0, 300, 600, 1200, 2500, or 5000 mg/kg body weight (BW). One male mouse in the 600 mg/kg BW dose group was observed as having rough hair; no animals died as a result of Automate Yellow 8 Petroleum administration. Treatment with the above doses produced polychromatic erythrocyte to red blood cell (PCE/RBC) ratios of 1.30, 1.27, 1.59, 1.67, 1.41, and 1.35%, respectively, in males and 1.57, 1.40, 1.44, 1.51, 1.48, and 0.96%, respectively, in females. The high dose of Automate Yellow 8 Petroleum was greater than 50% of the control PCE ratio (1.35% vs 1.30%, males; 0.96% vs 1.57%, females). Therefore, this dose was selected as the high dose for the definitive assay. No other statistical evaluations of the range-finding data were deemed necessary.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
On the basis of the results, it is concluded that Automate Yellow 8 Petroleum does not induce MN in polychromatic erythrocytes from swiss-Webster mice under the conditions of this assay.

Executive summary:

The peripheral erythrocyte micronucleus (MN) assay was used to evaluate the clastogenic potential of Automate Yellow 8 Petroleum. Doses of 0, 1200, 2500, and 5000 mg/kg BW of Automate Yellow 8 Petroleum in males produced PCE/RBC values of 1.68, 1.80, 1.58, and 1.62%, respectively. In females, the same doses produced PCE/RBC values of 1.58, 1.50, 1.37, and 1.54%, respectively. In males, doses of 1200, 2500, and 5000 mg/kg BW yielded 0.06, 0.22, and 0.16% PCE with MN, respectively, compared with a vehicle control value of 0.20%. In females, the same doses yielded 0.22, 0.20, and 0.24% PCE with MN, respectively, compared with a vehicle control value of 0.26%. In contrast, benzene yielded 3.90% PCE with MN in male mice. None of the doses of Automate Yellow 8 Petroleum induced a statistically significant increase in the micronucleus frequency.

On the basis of the results, it is concluded that Automate Yellow 8 Petroleum does not induce MN in polychromatic erythrocytes from swiss-Webster mice under the conditions of this assay.