Bis(4-chlorophenyl) sulphone

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:

Study initiation date: February 1st, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Valid study according to generally accepted protocol, comparable to guideline study under GLP conditions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Reference 2

Reference Type:

other: cover letter & Attachment to study report

Title:

CHO/HGPRT Mutation Assay With Confirmation (Final Report) With Attachment And Cover Letter Dated 111491

Author:

N.N.

Year:

1991

Bibliographic source:

EPA/OTS; Doc #86-920000116

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Heddle (1973) and Heddle et al (1983)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Method references:
- Heddle JA (1973). A rapid in vivo test for chromosomal damage. Mutation Res 18:187-90.
- Heddle JA, Hite M, Kirkhart B, Mavournin K, MacGregor JT, Newell GW & Salamone M (1983). The induction of micronuclei as a measure of genotoxicity. A report of the U. S. Environmental Protection Agency Gene-Tox Program. Mutation Res 123:61-118.
- Kastenbaum MA & Bowman KO (1970). Tables for determining the statistical significance of mutation frequencies. Mutation Res 9:527-49
- Mackey BE & MacGregor JT (1979). The Micronucleus test; statistical design and analysis. Mutation Res. 64:195-204.
- Wahnschaffe U, Bitsch A, Kielhorn J & Mangelsdorf I (2005). Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test. Journal of Carcinogenesis 4:3 doi:10.1186/1477-3163-4-3.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(4-chlorophenyl) sulphone

EC Number:

201-247-9

EC Name:

Bis(4-chlorophenyl) sulphone

Cas Number:

80-07-9

Molecular formula:

C12H8Cl2O2S

IUPAC Name:

1-chloro-4-(4-chlorobenzenesulfonyl)benzene

Details on test material:

- Name of test material (as cited in study report): 4,4'-dichlorodiphenylsulfone
- Substance type: organic
- Physical state: White powder
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: not provided to the laboratory
- Storage condition of test material: Room temperature, protected from light
- Other: Test item sample received 1989-01-02

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley Inc, Frederick, MD, U.S.A.
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males 26-35 g, females 22-31 g
- Assigned to test groups randomly: yes
- Housing: in an AAALAC-accredited facility
- Acclimation period: 13 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 74±6 °F = 23.3±3.4 °C
- Humidity (%): 50±20
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Corn oil was used as carrier vehicle for the test item.

Details on exposure:

Intraperitoneal, single dose application

Duration of treatment / exposure:

24, 48, and 72 h after treatment

Frequency of treatment:

Once

Doses / concentrations

Remarks:

Doses / Concentrations:
196, 980 and 1960 mg/kg bw
Basis:
other: actual injected

No. of animals per sex per dose:

5 (13 experimental groups of five males and five females)

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Route of administration: injected IP
- Doses / concentrations: dose level of 0.25 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow cells were microscopically examined for micronucleated polychromatic erythrocytes.
Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei.
The proportion of polychromatic erythrocytes to total erythrocytes and the number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes were also enumerated.

Details of tissue and slide preparation:

At the scheduled sacrifice time, five mice per sex were sacrificed by Carbon Dioxide asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL FBS. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted. Slides were coded using a random number table by an individual not-involved with the scoring process.

Evaluation criteria:

Criteria for Determination of a Valid Test:
The mean incidence of micronucleated polychrormatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the negative (vehicle) control.
The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the negative control (p ≤ 0.05, Kastenbaum-Bowman Tables).
The positive response must be dose-dependent or must be observed at a single dose level at adjacent sacrifice times.
If a single treatment group is significantly elevated at one sacrifice time, the assay is considered a suspect or unconfirmed positive and a repeat assay will be recommended.

Statistics:

Statistical significance will be determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. The test article is considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes is observed relative to the vehicle control (p ≤ 0.05, Kastenbaum-Bowman Tables)

Results and discussion

Test results

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:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000, 1500, 2000, 2500 or 5000 mg test article/kg body weight
- LD50 2448 mg/kg bw, (see 7.2.4 for details)

RESULTS OF DEFINITIVE STUDY
- no increase in mortality
- clinical signs of toxicity included slight head tilt, lethargy, piloerection, hunched posture and prostration.
- Induction of micronuclei (for Micronucleus assay): no statistical significant increase (p>0.05) at any dose level or collection time.
- Ratio of PCE/NCE: no apparent reduction in the PCE/NCE ratio.
- positive control TEM induced a significant increase in micronucleated polychromatic erythrocytes in males and females

Any other information on results incl. tables

Table 2

Treatment	Sex	Time [h]	Number of mice	PCE/Total Erythrocytes	Number per 1000 PCE’s (mean±SD)	Number per PCE’s scored
Corn oil 10 mL/kg	M	24	5	0.44	0.0±0.00	0 / 5000
		48	5	0.59	0.8±0.84	4 / 5000
		72	5	0.53	1.2±1.30	6 / 5000
	F	24	5	0.52	0.6±0.55	3 / 5000
		48	5	0.53	0.8±0.84	4 / 5000
		72	5	0.59	0.0±0.00	0 / 5000
DCDPS 196 mg/kg	M	24	5	0.45	1.2±1.64	6 / 5000
		48	5	0.53	2.2±1.10	11 / 5000
		72	5	0.48	0.0±0.00	0 / 5000
	F	24	5	0.56	1.8±1.30	9 / 5000
		48	5	0.53	1.8±1.30	9 / 5000
		72	5	0.60	0.2±0.45	1 / 5000
DCDPS 980 mg/kg	M	24	5	0.45	1.2±0.84	6 / 5000
		48	5	0.48	2.6±1.95	13 / 5000
		72	5	0.31	1.0±1.00	5 / 5000
	F	24	5	0.47	1.2±1.30	6 / 5000
		48	5	0.48	0.8±0.84	4 / 5000
		72	5	0.23	0.4±0.55	2 / 5000
DCDPS 1960 mg/kg	M	24	5	0.43	0.2±0.45	1 / 5000
		48	5	0.43	1.2±1.64	6 / 5000
		72	5	0.21	0.4±0.55	2 / 5000
	F	24	5	0.57	1.0±1.41	5 / 5000
		48	5	0.48	1.0±0.71	5 / 5000
		72	5	0.21	0.2±0.45	1 / 5000
Triethylenemelamine 0.25 mg/kg	M	24	5	0.48	41.8±0.11	209 / 5000*
	F	24	5	0.40	32.0±7.39	160 / 5000*

* (p ≤ 0.05, Kastenbaum-Bowman Tables)

For the toxicity study, DCDPS was administered by IP injection to male and female ICR mice at six treatment levels and a vehicle control: 500, 1000, 1500, 2000, 2500 or, 5000 mg test article/kg body weight which was administered in a total volume of 10 ml test article-vehicle mixture/kg body weight. The LD50 was calculated by probit analysis to be approximately 2448 mg/kg. The high dose for the micronucleus test was set at 1960 mg/kg which was estimated to be 80% of the LD50.

For the micronucleus test, male and female mice were dosed by a single IP injection of 196, 980, or 1960 mg/kg. No test substance related mortality was identified. Clinical signs of toxicity included slight head tilt, lethargy, piloerection, hunched posture, and prostration. Control animals appeared normal during the study.

The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes scored and the proportion of polychromatic erythrocytes per total erythrocytes are summarized and presented for each treatment group by sacrifice time in Table 2. No apparent reduction in the ratio of polychromatic erythrocytes to total erythrocytes in males or females was observed. The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was not statistically increased in males and females, regardless of dose level or bone marrow collection times (p>0.05, Kastenbaum-Bowman Tables). TEM induced a significant increase in micronucleated polychromatic erythrocytes in male and female mice relative to the vehicle control (p ≤ 0.05, Kastenbaum-Bowman Tables).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of this assay DCDPS gave an unequivocal negative, ie. non-mutagenic response after a single dose application.

Executive summary:

DCDPS was tested under GLP in a valid and conclusive study similar to the OECD TGD 474 (1997).

Groups of 5 male and 5 female ICR mice were exposed to 196, 980 or 1960 mg/kg of DCDPS which was administered as a single intraperitoneal (i.p.) injection. The high dose level was calculated to be 80% of the LD₅₀. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. No change in the ratio of polychromatic erythrocytes to total erythrocytes was observed in males or female mice in the test item treated groups. Clinical signs of toxicity were present at the highest dose level.

No significant increases in micronucleated polychromatic erythrocytes were observed at 24, 48 or 72 hours after dose administration in males or females. The results of the assay indicate that DCDPS did not induce a significant increase in micronucleated polychromatic erythrocytes in male or female ICR mice. DCDPS was concluded to be negative in the mouse erythrocyte micronucleus assay.