4-chlorophthalic anhydride

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:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Micronucleus

Test material

Test material information

Constituent 1

Reference substance name:

4-chlorophthalic anhydride

EC Number:

204-251-9

EC Name:

4-chlorophthalic anhydride

Cas Number:

118-45-6

Molecular formula:

C8-H3-Cl-O3

IUPAC Name:

5-chloro-1,3-dihydro-2-benzofuran-1,3-dione

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

The ICR mice were ordered from Harlan Sprague Dawley, Inc. and were approximately 6-8 weeks of age at study initiation, weighing 26.2-35.1 g (males) and 21.5-30.6 g (females). Up to five mice of the same sex were group housed in polycarbonate cages. The controlled environment parameters were 72 ± 3°F, 50 ± 20% relative humidity and a 12-hour light/dark cycle. The mice had free access to certified rodent chow and water.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Dimethyl sulfoxide (DMSO) was determined to be the vehicle of choice based on a solubility determination of 4-CLPA; however, DMSO was not compatible with this test system and corn oil was selected as an alternative vehicle. 4-CLPA formed a workable, cloudy, yellow suspension in corn oil at 100 mg/mL, the maximum concentration tested.

Details on exposure:

In the pilot toxicity assay, five male and five female mice were exposed to 4 CLPA at a dose of 2000 mg/kg and two male mice each to 1, 10, 100, or 1000 mg/kg. 4-CLPA dosing formulations were administered at a volume of 20 mL/kg by a single IP injection. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration and 1 and 3 days after dose administration.

For the toxicity assay, all mice were weighed immediately prior to dose administration and the dose volume was based on individual body weight. In the toxicity study, animals (5 animals/sex/group) were exposed to 100, 200, 400, 600 and 800 mg/kg 4-CLPA. 4-CLPA dosing formulations were administered at a volume of 20 mL/kg by a single IP injection. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration and 1 and 3 days after dose administration.

For the definitive micronucleus assay, there were seven groups, each containing 5 male and 5 female ICR mice. Animals in five of these groups were treated either with the controls (negative or positive) or with 4 CLPA at a dose of 12.5, 25 or 50 mg/kg and were euthanized 24 hours after treatment. Animals in the other two groups were treated either with the negative control or 4-CLPA at a dose of 50 mg/kg and were euthanized 48 hours after treatment. Additional replacement animals (5 animals/sex) were included in the high dose group, 50 mg/kg, to ensure that the availability of 5 animals/sex for micronucleus analysis. 4 CLPA vehicle mixture, the vehicle alone or the positive control was administered by a single IP injection at a dose volume of 20 mL/kg. All mice in the experimental and control groups were weighed immediately before dose administration and the dose volume was based on individual body weight. Mice were observed after dose administration for clinical signs of toxicity.

Frequency of treatment:

Single treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Toxicity study

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Toxicity study

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

Toxicity study

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Remarks:

Toxicity study

Doses / concentrations 5

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

Toxicity study

Doses / concentrations 6

Dose / conc.:

12.5 mg/kg bw/day (actual dose received)

Remarks:

Definitive study

Doses / concentrations 7

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

Definitive study

Doses / concentrations 8

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Definitive study

No. of animals per sex per dose:

Preliminary Assay: five male and five female mice were exposed to 4 CLPA at a dose of 2000 mg/kg and two male mice each to 1, 10, 100, or 1000 mg/kg
Toxicity Assay: 5 animals/sex/group
Definitive Assay: 5 animals/sex/group

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes, Cyclophosphamide monohydrate (CP, CAS number 6055-19-2)

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

At the scheduled sacrifice times, five mice per sex per dose were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. 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 pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May Gruenwald Giemsa and permanently mounted.

Bone marrow cells [polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)], were analyzed for the presence of micronuclei. Polychromatic erythrocytes are young, immature red blood cells that stain bluish while normochromatic erythrocytes or normocytes are mature red blood cells that stain pink. Micronuclei are round, darkly-staining nuclear fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei can occur in both PCEs (MPCEs) and NCEs (MNCEs).

To control for bias, slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification (10 x 40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10 x 100), 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:

To quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and dose group. As a guide to interpretation of the data, 4-CLPA was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p is less than or equal to 0.05, Kastenbaum Bowman Tables) at any sampling time. However, values that were statistically significant but did not exceed the range of historical negative or vehicle controls were judged as not biologically significant. 4-CLPA was judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and dose group. Statistical significance was determined using the Kastenbaum Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the pilot study, mortality was observed in 5/5 male mice and 5/5 female mice at 2000 mg/kg and 2/2 males at 1000 mg/kg. Clinical signs following dose administration included: piloerection in males at 10 and 100 mg/kg and lethargy in male mice at 100 mg/kg.

In the toxicity study, Mortality occurred within three days of dose administration as follows: 2/5 males and 2/5 females at 100 mg/kg, 4/5 males and 3/5 females at 200 mg/kg and all males and females at 400, 600 and 800 mg/kg. Clinical signs following dose administration included: piloerection, lethargy, and hunched position in male and female mice treated at 100, 200 and 400 mg/kg as well as in males at 600 mg/kg. Convulsions were seen in males and females at 100 and 200 mg/kg, in females at 600 mg/kg and in males and females at 800 mg/kg. Irregular breathing was observed in males at 200 mg/kg and in males and females at 800 mg/kg and ataxia in males and females at 400 mg/kg and in males at 600 mg/kg. In addition, prostration was observed in females at 600 mg/kg and in males and females at 800 mg/kg. Based upon these results, the high dose for the definitive micronucleus study was set at 50 mg/kg, which was estimated to be the maximum tolerated dose.

In the definitive micronucleus study, no mortality was observed in any male or female dosed at 12.5, 25 or 50 mg/kg. Clinical signs following dose administration included: piloerection and lethargy in males and females at 50 mg/kg. All other animals treated with the test article or controls appeared normal during the course of the study.

Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after treatment were examined microscopically for presence of micronuclei (MPCEs or MNCEs). Reductions, up to 18%, in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. The number of micronucleated polychromatic erythrocytes per 10,000 polychromatic erythrocytes in 4-CLPA-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose or bone marrow collection time (p is greater than 0.05, Kastenbaum-Bowman Tables).

In this study, all criteria for a valid test were met as specified in the protocol. CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p is less than 0.05, Kastenbaum-Bowman Tables). The negative and positive controls were consistent with the historical control data, indicating that there was no problem with the test system or the quality of the test.

Any other information on results incl. tables

Summary of Bone Marrow Micronucleus Analysis Following a Single Dose of 4-Chlorophthalic Anhydride (4-CLPA; CAS# 118-45-6) in ICR Mice

Treatment (20 mL/kg)	Sex	Time (hr)	Number of Mice	PCE/Total             Erythrocytes        (Mean +/- SD)	Change from Control (%)	Micronucleated Polychromatic Erythrocytes
						Number per 1000 PCEs (Mean +/- SD)	Number per PCEs Scored
Corn oil*	M	24	5	0.477 ± 0.05	---	0.6 ± 0.22	6 / 10000
	F	24	5	0.492 ± 0.06	---	0.3 ± 0.27	3 / 10000
4-CLPA
12.5 mg/kg	M	24	5	0.472 ± 0.02	-1	0.6 ± 0.42	6 / 10000
	F	24	5	0.464 ± 0.04	-6	0.4 ± 0.22	4 / 10000
25 mg/kg	M	24	5	0.438 ± 0.04	-8	0.5 ± 0.35	5 / 10000
	F	24	5	0.432 ± 0.07	-12	0.5 ± 0.35	5 / 10000
50 mg/kg	M	24	5	0.476 ± 0.06	0	0.6 ± 0.42	6 / 10000
	F	24	5	0.401 ± 0.04	-18	0.6 ± 0.22	6 / 10000
CP*
50 mg/kg	M	24	5	0.421 ± 0.05	-12	25.8 ± 4.07	*258 / 10000
	F	24	5	0.405 ± 0.04	-18	24.4 ± 4.11	*244/ 10000
Corn oil*	M	48	5	0.556 ± 0.05	---	0.5 ± 0.35	5 / 10000
	F	48	5	0.485 ± 0.00	---	0.9 ± 0.22	9 / 10000
4-CLPA
500 mg/kg	M	48	5	0.483 ± 0.03	-13	0.5 ± 0.35	5 / 10000
	F	48	5	0.451 ± 0.02	-7	0.5 ± 0.00	5 / 10000
*Statistically significant, p less than or equal to 0.05 (Kastenbaum‑Bowman Tables)

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 24 Hours Following a Single Dose of 4-Chlorophthalic Anhydride(4-CLPA; CAS# 118-45-6) in ICR Mice

Treatment (20 mL/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Corn oil*	M	101	0.461	1 / 2000
		102	0.542	1 / 2000
		103	0.511	1 / 2000
		104	0.435	2 / 2000
		105	0.438	1 / 2000
	F	106	0.435	1 / 2000
		107	0.538	1 / 2000
		108	0.438	1 / 2000
		109	0.489	0 / 2000
		110	0.561	0 / 2000
4-CLPA
12.5 mg/kg	M	111	0.488	1 / 2000
		112	0.491	2 / 2000
		113	0.467	1 / 2000
		114	0.467	0 / 2000
		115	0.448	2 / 2000
	F	116	0.493	1 / 2000
		117	0.508	1 / 2000
		118	0.420	1 / 2000
		119	0.412	0 / 2000
		120	0.488	1 / 2000
25 mg/kg	M	121	0.412	1 / 2000
		22	0.492	2 / 2000
		123	0.408	1 / 2000
		124	0.417	0 / 2000
		125	0.462	1 / 2000
	F	126	0.507	1 / 2000
		127	0.336	1 / 2000
		128	0.404	1 / 2000
		129	0.446	0 / 2000
		130	0.466	2 / 2000
50 mg/kg	M	131	0.438	0 / 2000
		132	0.438	1 / 2000
		133	0.428	1 / 2000
		134	0.521	2 / 2000
		135	0.556	2 / 2000
	F	136	0.381	1 / 2000
		137	0.451	1 / 2000
		138	0.417	2 / 2000
		139	0.341	1 / 2000
		140	0.413	1 / 2000
CP* 50 mg/kg	M	141	0.457	45 / 2000
		142	0.443	56 / 2000
		143	0.353	53 / 2000
		144	0.467	42 / 2000
		145	0.383	62 / 2000
	F	146	0.331	44 / 2000
		147	0.431	62 / 2000
		148	0.425	41 / 2000
		149	0.431	51 / 2000
		150	0.406	46 / 2000

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 48 Hours Following a Single Dose of 4-Chlorophthalic Anhydride(4-CLPA; CAS118-45-6) in ICR Mice

Treatment (20 mL/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Corn oil*	M	151	0.570	1 / 2000
		152	0.592	0 / 2000
		153	0.601	1 / 2000
		154	0.468	1 / 2000
		155	0.551	2 / 2000
	F	156	0.488	1 / 2000
		157	0.483	2 / 2000
		158	0.486	2 / 2000
		159	0.481	2 / 2000
		160	0.485	2 / 2000
4-CLPA
50 mg/kg	M	161	0.464	1 / 2000
		162	0.462	1 / 2000
		163	0.536	2 / 2000
		164	0.457	0 / 2000
		165	0.495	1 / 2000
	F	166	0.441	1 / 2000
		167	0.480	1 / 2000
		168	0.437	1 / 2000
		169	0.453	1 / 2000
		170	0.443	1 / 2000

Applicant's summary and conclusion

Conclusions:

The test substance was negative in a micronucleus assay.

Executive summary:

Genetic toxicity was determined in an in vivo mammalian erythrocyte micronucleus test performed according to OECD 474 and in compliance with GLP criteria. In this study, 5 male and 5 female ICR mice per dose were treated once with test concentrations of 12.5, 25 and 50 mg/kg body weight, administered intraperitoneal 20 mL/kg. These test concentrations were based on a pilot toxicity assay. After dose administration, mice were observed for clinical signs of toxicity. Furthermore, body weights were determined before treatment. The test animals were sacrificed 24 hours after treatment and subsequently the bone marrow was isolated. After slide preparation, 2000 polychromatic erythrocytes were scored per animal for the presence of micronuclei. No significant increase in micronucleated polychromatic erythrocytes was observed at any of the tested concentrations. A single intraperitoneal administration of test substance at doses up to 100 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Based on these test results, it is concluded that the test substance was negative in the micronucleus test.