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Toxicological information

Genetic toxicity: in vivo

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out under GLP conditions and in accordance with OECD Guideline for Testing of Chemicals No. 474 (1997) without restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Chloroform
- Physical state: colourless, volatile liquid
- Analytical purity: 99.98 %
- Lot/batch No.: R863 22/07/2008
- Expiration date of the lot/batch: 16 October 2008
- Stability under test conditions: yes
- Storage condition of test material: refrigerated at 2-8 °C in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, UK
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 183 to 316 g for males; 172 to 225 g for females
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: up to six of the same sex in cages
- Diet (e.g. ad libitum): access ad libitum to SQC Rat and Mouse Maintenance Diet No. 1, Expanded (Special Diets Services Ltd., Witham)
- Water (e.g. ad libitum): Mains water provided ad libitum
- Acclimation period: 5 to 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The test article was dissolved in corn oil
Details on exposure:
The test article and vehicle were weighed into suitable containers and added to a designated dose pot and stirred until mixed to make the final volume. A two hour expiry was added to each formulation following dissolution. The dosing preparations had concentrations of 12, 24 and 48 mg/mL. A total of 10 mL of each dosing preparation was administered by oral gavage to test animals.
Duration of treatment / exposure:
5 days
Frequency of treatment:
Once daily
Post exposure period:
24 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
120 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
240 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
480 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
6 males, 6 females
Control animals:
yes, concurrent vehicle
Positive control(s):
The clastogenic positive control was cyclophosphamide (CPA, Sigma Chemical Co, Poole, UK) freshly prepared in saline. The aneugenic positive control was carbendazim (CBZ Sigma Chemical Co, Poole, UK) freshly prepared in 1 % (w/v) aqueous methylcellulose (1 % MC).

Examinations

Tissues and cell types examined:
Body weights were recorded on each day of dosing and on the post-dose observation day. Body temperatures were recorded approximately 2 hours following the doses on Day 1 and Day 5. Blood samples were taken from satellite animals dosed by the same route, dose levels and at the same dosing frequency as that described for the micronucleus animals.
Details of tissue and slide preparation:
Test article, vehicle and CBZ-treated rats were sampled in groups, 24 hours after the final administration; CPA treated rats were sampled 24 hours after a single dose. Rats were killed by an overdose of sodium pentobarbitone, given via intraperitoneal injection and subsequently ensured by cervical dislocation, in the same sequence used for dosing.
One femur (Range Finder Experiment) or both femurs (Micronucleus Experiment) were exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes.
The bone marrow suspensions from the Range Finder animals and one set of suspensions (from one femur per animal) from the Micronucleus Experiment were processed and slides prepared as follows:
A further 3 mL of foetal bovine serum was added to the tubes, which were then centrifuged at 200 g for approximately five minutes; the serum was aspirated to leave one or two drops and the cell pellet. It should be noted that a second washing step was performed (resuspending the cell pellet in 3 mL of serum), where considered necessary to optimise slide quality.
The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of two slides labelled with the appropriate study number, sampling time, sex, date of preparation and animal number. The latter served as the code so analysis could be conducted "blind". A smear was made from the drop by drawing the end of a clean slide along the labelled slide.
Slides were allowed to air dry and then fixed for 10 minutes in absolute methanol. Slides were allowed to dry (and stored at room temperature until required for staining). Staining was performed on the same day as slide preparation. One slide from each set was taken (any remaining slides were kept in reserve). Prior to staining any stored slides were fixed again for 10 minutes in absolute methanol. After rinsing several times in distilled water, slides were stained for 5 minutes in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then allowed to dry and stored in the dark at room temperature prior to analysis.
Additional spare slides from female animals were stained and analysed from animal numbers 990 (vehicle), 955 (120 mg/kg/day), 966 (240 mg/kg/day), 970, 976, 967 (CPA), and 981 (Carbendazim, 1500 mg/kg/day). This was due to sub-optimal staining of initial slides.
In order to allow optimum slide preparation for potential antikinetochore (AK) staining, bone marrow collected from the second femur per animal was passed through a cellulose filtration system to remove 60%-70% of the nucleated cell fraction. This procedure is outlined below:
Bone marrow was sampled as per previously described into 2 mL foetal bovine serum into appropriately labelled centrifuge tubes. An additional 4 mL of serum was added to each bone marrow sample prior to adding to pre-prepared cellulose filtration columns.
The cellulose columns contained 2 mL of an equal mix of type 50 and  cellulose achieving a 50 mg/mL solution. These were prepared the day prior to filtration. Preparation of the columns was in accordance to SOP MT 45/030 and as recommended by published data [ ].
Once filtered the bone marrow cells were centrifuged at 200 x ‘g’ for 5 minutes at room temperature and standard smears prepared. Two to three slides were prepared per animal in this way depending on the quantity of bone marrow available.
Following fixation, prepared slides were stored in slide boxes at nominal 20C prior to possible further AK staining.
Due to the negative micronucleus data obtained from this study, further mechanistic analysis was not required. As such, filtered slides were not processed further.
Evaluation criteria:
The assay was considered valid if all the following criteria were met:
1. The incidence and distribution of MN PCE in vehicle control groups were consistent with the laboratory’s historical vehicle control data, and
2. The proportion of immature erythrocytes among total erythrocytes (expressed as %PCE) should not be less than 20% of the control value at each test article dose, and
3. At least five animals (per sex) out of each group (males and females) were available for analysis, and
4. The positive control chemical (CPA) induced a statistically significant increase in the frequency of MN PCE.
Acceptance under any other criteria are discussed in the results section.

For valid data, the test article was considered to induce micronuclei if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data
3. A dose-response trend in the proportion of MN PCE was observed (where more than two dose levels were analysed).
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were to be dealt with on a case by case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result [ ]. Biological relevance was taken into account, for example consistency of response within and between dose levels.
Statistics:
After completion of microscopic analysis and decoding of the data the following were calculated:
1. % PCE for each animal and the mean for each group. The group mean % PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity
2. Frequency of MN PCE (i.e. MN per 2000 PCE) and % MN PCE for each animal and the group mean % MN PCE (+/- standard deviation).
The numbers of MN PCE in vehicle control animals were compared with the laboratory's historical control data to determine whether the assay was acceptable. For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square test.
The numbers of micronucleated PCE in each treated group (males and females separately) were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square. Probability values of p < 0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
As the heterogeneity chi-square test provided evidence of significant (p < 0.05) variability between animals within at least one group, non-parametric analysis by use of the Wilcoxon rank sum test was performed. This was conducted on male and female data.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 240 and 480 mg/kg body weight
- Clinical signs of toxicity in test animals: Clinical signs of toxicity in the 240 mg/kg dose group included decreased activity, piloerection, ptosis, mouth rubbing, paddling and hunched posture and similar effects plus eye-closure, ataxia and unkempt appearance at 480 mg/kg, which persisted longer than in the lower dose group.
- Evidence of cytotoxicity in tissue analyzed: Rats treated with chloroform showed group mean PCE/NCE ratios (expressed as % PCE) that decreased in a dose dependent manner, with the highest dose exhibiting 38 % PCE (males) or 27 % PCE (females). These ratios were markedly lower than the concurrent vehicle control values of 62 % or 44 % PCE (males and females respectively). These data were also lower than historical values and as such were considered to represent some evidence of bone marrow toxicity and target organ exposure.
- Rationale for exposure: Data from a bone marrow chromosome aberration study indicated statistically significant increases in structural chromosome aberrations following five days of oral exposure
- Harvest times: The duration of the study has been shown to be of sufficient duration for the expression of any genotoxic potential.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE
- Ratio of PCE/NCE (for Micronucleus assay): The polychromatic erythrocytes to normal erythrocytes ratio of negative (vehicle) control male rats was slightly higher than that of the historical vehicle control (normal) range (62 % PCE versus 39 to 59 %). The ratio was within normal values for control females. As individual frequencies of micronucleated PCE were consistent with historical vehicle control distribution data (both genders) and vehicle treated animals did not exhibit clinical signs of toxicity, the vehicle data were accepted as valid. The clastogen cyclophosphamide positive control group exhibited significantly increased numbers of micronuleated PCE, which was also seen in the aneugenic Carbendazim positive control group.
- Appropriateness of dose levels and route: The results of the bioanalysis confirm that animals dosed at 120, 240 and 480 mg/kg body weight were systemically exposed to chloroform
- Statistical evaluation:
1. The incidence and distribution of MN PCE in the vehicle control group were consistent with the laboratory's historical vehicle control data. This was apparent for female animals but not for males where the group mean %PCE slightly exceeded historical values (62% PCE versus 39%-59% range). The reason for this high value was not clear but may have been due to staining characteristics. As this increase was small, with individual frequencies of MN PCE consistent with historical vehicle control distribution data, with animals showing no clinical observations of ill health, these data were accepted as valid.
2. The proportion of immature erythrocytes among total erythrocytes (expressed as %PCE) were not less than 20% of the control value at each test article dose analysed.
3. At least five animals out of each group (male and female) were available for analysis.
4. The positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE (Appendix 6).
The assay data were therefore considered valid.

Any other information on results incl. tables

Table 1: Individual animal micronucleus frequencies - males

Treatment

(mg/kg/day)

Animal

Number

PCE

Count

NCE

Count

%

PCE

Total PCE

Count

MN

PCE

 %

MN PCE

Vehicle

469

638

362

63.80

2000

5

0.25

467

525

475

52.50

2000

1

0.05

456

736

264

73.60

2000

0

0.00

473

482

518

48.20

2000

0

0.00

452

642

358

64.20

2000

1

0.05

465

721

279

72.10

2000

1

0.05

120

458

657

343

65.70

2000

3

0.15

450

846

154

84.60

2000

3

0.15

468

610

390

61.00

2000

1

0.05

471

706

294

70.60

2000

3

0.15

447

623

377

62.30

2000

2

0.10

437

425

575

42.50

2000

1

0.05

240

454

401

599

40.10

2000

1

0.05

451

493

507

49.30

2000

1

0.05

475

590

410

59.00

2000

2

0.10

463

433

567

43.30

2000

0

0.00

476

571

429

57.10

2000

1

0.05

445

458

542

45.80

2000

1

0.05

480

464

522

478

52.20

2000

1

0.05

462

405

595

40.50

2000

1

0.05

474

164

836

16.40

2000

4

0.20

470

Data Omitted*

466

334

666

33.40

2000

0

0.00

442

486

514

48.60

2000

4

0.20

CPA, 20+

472

461

539

46.10

2000

34

1.70

446

620

380

62.00

2000

53

2.65

461

408

592

40.80

2000

23

1.15

457

401

599

40.10

2000

41

2.05

443

490

510

49.00

2000

47

2.35

459

490

510

49.00

2000

29

1.45

+ administered as a single dose; * animal killed in extremis, data omitted; MN micronucleated

Table 2: Individual animal micronucleus frequencies - females

Treatment

(mg/kg/day)

Animal

Number

PCE

Count

NCE

Count

%

PCE

Total PCE

Count

MN

PCE

 %

MN PCE

Vehicle

968

397

603

39.70

2000

0

0.00

992

427

573

42.70

2000

4

0.20

995

470

530

47.00

2000

3

0.15

990

443

557

44.30

2000

2

0.10

979

410

590

41.00

2000

4

0.20

959

482

518

48.20

2000

1

0.05

120

973

417

583

41.70

2000

2

0.10

963

445

555

44.50

2000

2

0.10

955

421

579

42.10

2000

2

0.10

977

337

663

33.70

2000

3

0.15

980

447

553

44.70

2000

2

0.10

965

459

541

45.90

2000

0

0.00

240

988

338

662

33.80

2000

0

0.00

986

399

601

39.90

2000

0

0.00

975

436

564

43.60

2000

3

0.15

966

375

625

37.50

2000

0

0.00

984

460

540

46.00

2000

0

0.00

971

427

573

42.70

2000

0

0.00

480

982

172

828

17.20

2000

3

0.15

962

371

629

37.10

2000

2

0.10

969

399

601

39.90

2000

7

0.35

994

152

848

15.20

2000

3

0.15

974

230

770

23.00

2000

1

0.05

991

271

729

27.10

2000

4

0.20

CPA, 20+

970

297

703

29.70

2000

34

1.70

993

219

781

21.90

2000

18

0.90

996

339

661

33.90

2000

22

1.10

976

352

648

35.20

2000

22

1.10

964

257

743

25.70

2000

13

0.65

967

188

812

18.80

2000

31

1.55

+ administered as a single dose; MN micronucleated

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Chloroform did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female rats treated up to 480 mg/kg body weight/day for five consecutive days, under the experimental conditions employed.
Executive summary:

Chloroform was tested for its ability to induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female young adult Sprague Dawley rats, following 5 days of repeat oral dosing. The test was carried out under GLP conditions and in accordance with OECD Guideline No. 474. From range finding tests, 480 mg/kg body weight/day was considered a suitable maximum tolerated dose under the assay conditions. The final micronucleus study was performed by administration by oral gavage of 120, 240 or 480 mg/kg body weight/day chloroform in corn oil for five consecutive days to groups of six male and six female rats, respectively. Analyses of formulations administered to animals demonstrated variability in terms of achieved concentrations from all of the sampling points across the range of concentrations used and most particularly at the low dose level (-9.9 to -91.4 % of the nominal concentration of 12 mg/mL; -4.3 to -36.4 % of nominal 24 mg/mL; -2.7 to -20.3 % of nominal 48 mg/mL used for dosing). However, the analyses of blood plasma confirmed that animals were systemically exposed to chloroform with increasing exposure with both concentration and time. Two additional groups of six males and six females were treated once with a clastogen positive control, cyclophosphamide, 24 hours prior to necropsy (20 mg/kg). Two groups of six males and six females were treated twice with an aneugenic positive control, Carbendazim (1500 mg/kg and 2000 mg/kg). During the treatment period, clinical signs observed essentially in the 480 mg/kg/day group included among others ataxia, hunched posture, hypothermia, lethargy, decreased activity, ptosis, piloerection and tremors. The group mean frequency of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE), ratio expressed as % PCE, of the negative vehicle controls were considered as valid, although negative male control rats exhibited a slightly increased ratio. The positive control groups exhibited significantly increased frequencies of micronucleated PCE in comparison with the concurrent controls. The assay system was therefore considered as valid. Rats treated with chloroform showed group mean % PCE values that decreased in a dose dependent manner, with the highest dose group (480 mg/kg/day) exhibiting 38 % PCE (males) or 27 % PCE (females). These were markedly lower than the concurrent vehicle control values of 62 % or 44 % PCE (male and females respectively) and also lower than historical values and as such were considered to represent some evidence of bone marrow toxicity and target organ exposure. The group mean frequencies of micronucleated PCE observed in the groups treated with chloroform were not significantly different to the vehicle controls. In addition, individual frequencies of micronucleated PCE were generally similar to those seen in the vehicle control groups and consistent with the laboratory's historical control distribution data. From the results of the study it is concluded that chloroform did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female Sprague Dawley rats treated up to 480 mg/kg body weight/day for five consecutive days, under the experimental conditions employed. Thus, chloroform was not genotoxic in the present micronucleus assay.