1-bromobutane

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:

18 September 1997 to 11 November 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Test material

Test material information

Test material form:

liquid

Details on test material:

n-Butyl bromide, batch number 7-014-1, was a clear colourless liquid. It was received on 11 August 1997 and stored at room temperature in the dark. Purity was stated as 99.13% and expiry date as August 1998.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

An excess number of out-bred CD-1 mice was obtained from Charles River UK Ltd, Margate, UK.
The micronucleus test in the mouse has been used in a number of collaborative trials, and correlates quite well with other systems as long as high enough doses and multiple sampling times are used.

Sex:

male/female

Details on test animals or test system and environmental conditions:

An excess number of out-bred CD-1 mice was obtained from Charles River UK Ltd, Margate, UK. They were housed in groups of the same sex in appropriate caging, cleaned and dried before use. Housing was in groups of no more than three animals for the main study and no more than four, for the range-finder. Bottled mains (public supply) water and diet (Special Diets Services Ltd, RMl.[E].SQC.) were provided ad libitum. These are routinely monitored and are not known to contain any biological or chemical entity which might interfere with the test system.
During the period of the study, the holding room was illuminated continuously by fluorescent light for 12 hours out of each 24 hour cycle and is designed to receive at least 15 fresh air changes per hour.
All mice were identified by numbered ear tag. Prior to the main study, male and female mice were randomised to groups of five using a system of randomly generated numbers. Checks were made on the first day of treatment to ensure group weights differed from the overall mean by no more than 5%. Excess animals were humanely killed.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Dosing preparations were made by suspending n-butyl bromide in corn oil to give the top concentrations.

Details on exposure:

Dilutions were made using corn oil. The test article preparations were protected from light and used within two hours of initial formulation.
Animals were weighed before dosing and the volume of vehicle, test article preparation or positive control solution to be administered was calculated based on a dose volume of 20 mL/kg. Animals were not starved prior to dosing. All treatments were given intraperitoneally.

Duration of treatment / exposure:

Range-finder: two consecutive days (survival permitting).
Main study: two consecutive days

Frequency of treatment:

Range-finder and Main study: once daily

Post exposure period:

Range-finder: 4 days
Main study: 24 hours after second dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

156.3 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

312.5 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

625 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 250 mg/kg bw/day

No. of animals per sex per dose:

Control groups: 10 (5 males/5 females)
156.3 mg/kg/day: 5 males
312.5 and 625 mg/kg/day: 10 (5 males/5 females)
1250 mg/kg/day: 5 females

Control animals:

yes

Positive control(s):

Cyclophosphamide (CPA, Sigma Chemical Co, Poole, UK) was freshly dissolved in physiological saline at 2 mg/mL to serve as the positive control.

Examinations

Tissues and cell types examined:

Slides from the CPA-treated mice were initially checked as described below to ensure the system was operating satisfactorily. The slides from all control and dose groups were arranged in numerical order by sampling time and sex and analysed by a person not connected with the dosing phase of the study.
Initially the relative proportions of polychromatic erythrocytes (PCE), seen as pale blue or blue/grey enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller yellow/orange-stained enucleate cells, were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed.
Counting continued (but of PCE only) until at least 2000 PCE had been observed.
All PCE containing micronuclei observed during these two phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded to a maximum of six per 2000 cells scored.
Slide analysis was performed off-site by an analyst trained in accordance with Covance Standard Operating Procedures. All slides and raw data have been returned to Covance for archiving in accordance with the archive statement in the report.

Details of tissue and slide preparation:

Test article and vehicle treated mice were killed in groups, 24 hours after the second administration; CPA-treated mice were killed 24 hours after the single dose. Mice were killed by asphyxiation with carbon dioxide (subsequently ensured by cervical dislocation) in the same order as they were dosed.
Both femurs from each animal 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 1 mL foetal bovine serum into appropriately labelled centrifuge tubes (one per animal).
The tubes were centrifuged (1250 x 'g', 2-3 minutes) and the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube 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 tag number. The latter served as a 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 were fixed for 5 minutes in absolute methanol before being stained according to the modification of Gollapudi and Kamra. One slide from each set of two was then taken, the other was kept in reserve. After rinsing several times in water, slides were stained for 10 minutes in filtered Giemsa stain diluted 1:6 (v/v) in distilled water. Stained slides were rinsed, and allowed to dry thoroughly before clearing in xylene for 3 minutes. When dry, the slides were mounted with coverslips.

Evaluation criteria:

The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the frequency of micronucleated PCE occurred at least at one dose, and
2) the frequency of micronucleated PCE at such a point exceeded the historical vehicle control range.

Statistics:

After completion of microscopic analysis and decoding of the data, the ratio of PCE/NCE for each animal and the mean for each group was calculated.
The individual and group mean frequency of micronucleated PCE/1000 were also determined.
PCE/NCE ratios were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity.
The group mean frequencies of micronucleated PCE in vehicle control animals were compared with historical negative control ranges to determine whether or not the assay was acceptable. For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity X2 test.
The numbers of micronucleated PCE in each treated group (males and females, separately and combined) were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine X2. Probability values of p ≤0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose response relationships.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

625 mg/kg/day (males) and 1250 mg/kg/day (females)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Selection of doses for main study
A difference was apparent in response between males and females. Maximum doses of 625 mg/kg/day (males) and 1250 mg/kg/day (females) were chosen for the main study.

Micronucleus assay
Validity of study
1) the incidence of micronucleated PCE in vehicle control groups fell within the historical vehicle control range, and
2) at least eight animals (males plus females) out of each group at each kill time were available for analysis, and
3) the positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE

Analysis of data
Groups of mice treated with n-butyl bromide exhibited PCE/NCE ratios which were similar to vehicle controls at both sampling times. Group mean frequencies of micronucleated PCE were also similar to those seen in vehicle control groups and were not significantly different by X2 analysis.

Any other information on results incl. tables

Selection of doses for main study

 The range-finder study was performed with the following results:

Dose (mg/kg/day)	Deaths	Observations
500	0 (females not dosed)	Lethargy, abnormal gait, piloerection, tremors, eye closure, sunken eyes.
750	1 males and 0 females	Lethargy, hunched posture, abnormal gait, piloerection, tremors, eye closure.
1000	2 males and 0 females	Lethargy, prostration, hunched posture, abnormal gait, piloerection, tremors, coldness, eye closure.
1250	3 males and 0 females	Lethargy, abnormal gait, piloerection, tremors, eye closure.
1500	3 males and 1 female	Lethargy, prostration, hunched posture, abnormal gait, piloerection, tremors, abnormal breathing, eye closure, sunken eyes. Tilted head, hyperactivity.
2000	3 males and 3 females	Lethargy, prostration, hunched posture, abnormal gait, piloerection, tremors, coldness, abnormal breathing, eye secretion, eye closure.

 

Main Study

Clinical Signs

Dose (mg/kg/day)	Sex	Observations
156.3	Males	Eye secretion, eye closure.
312.5	Males Females	Eye secretion, eye closure, lethargy. Eye closure.
625	Males Females	Eye secretion, eye closure, lethargy. Eye closure.
1250	Females	Eye secretion, eye closure, lethargy, abnormal gait, piloerection

 

Summary of group mean data

Data from n-butyl bromide

Treatment group (mg/kg/day)	Sex	Mean ratio PCE/NCE	Group mean frequency of micronucleated PCE (per 1000)
Vehicle control	Male	1.03	0.30
	Female	1.50	0.90
156.3	Male	0.99	0.40
312.5	Female	1.13	0.10
312.5	Male	1.22	0.20
625	Female	0.96	0.40
625	Male	0.95	0.90
1250	Female	1.05	0.40
CPA, 80+	Male	1.43	9.20
	Female	1.18	8.40

+ Administered as a single dose

 

Total group weights

Data for n-butyl bromide

Treatment (mg/kg/day)		Kill time after treatment (hours)
		24
		Male	Female
Vehicle control	Group number Total group weight (g)	1 146	7 116
156.3	Group number Total group weight (g)	2 144	-
312.5	Group number Total group weight (g)	3 145	8 121
625	Group number Total group weight (g)	4 145	9 119
	Group number Total group weight (g)	6* 144	-
1250	Group number Total group weight (g)	-	10 116
	Group number Total group weight (g)	-	12* 118
CPA, 40+	Group number Total group weight (g)	5 146	11 119

Group weights checked on the first day of dosing to ensure weights of all groups differ from mean by no more than 5%.

+ Administered as a single dose

*Included to be sampled in the event of deaths among animals in groups 4 (males) or 10 (females)

Mean group weight: males 145 g; females 118 g

 

Body weights and dosages

Data for n-butyl bromide

			Day 1		Day 2
Treatment (mg/kg/day)	Group/sex	Animal number	Body weight (g)	Dose given (mL)	Body weight (g)	Dose given (mL)
Vehicle control	1M	716 730 708 707 704	28 27 33 28 30	0.56 0.54 0.66 0.56 0.60	28 27 33 27 30	0.56 0.54 0.66 0.54 0.60
156.25	2M	717 711 729 710 713	27 28 31 29 29	0.54 0.56 0.62 0.58 0.58	28 28 32 28 29	0.56 0.56 0.64 0.56 0.58
312.5	3M	706 720 714 702 721	31 28 30 28 28	0.62 0.56 0.60 0.56 0.56	32 29 30 28 29	0.54 0.58 0.60 0.56 0.58
625	4M	719 722 705 718 703	30 29 30 27 29	0.60 0.58 0.60 0.54 0.58	30 29 28 27 27	0.60 0.58 0.56 0.54 0.54
	6M	728 725 715 712 709	27 30 30 27 30	0.54 0.60 0.60 0.54 0.60	29 30 29 28 29	0.58 0.60 0.58 0.56 0.58
CPA, 40+	5M	723 727 701 726 724	28 31 27 29 31	- - - - -	29 32 28 29 31	0.58 0.64 0.56 0.58 0.62
Vehicle control	7F	755 745 744 739 742	25 24 21 23 23	0.50 0.48 0.42 0.46 0.46	25 24 21 22 23	0.50 0.48 0.42 0.44 0.46
312.5	8F	750 752 759 751 746	22 23 25 25 26	0.44 0.46 0.50 0.50 0.52	23 23 25 24 25	0.46 0.46 0.50 0.48 0.50
625	9F	736 733 735 741 731	26 22 24 24 23	0.52 0.44 0.48 0.48 0.46	25 22 25 25 23	0.50 0.44 0.50 0.50 0.46
1250	10F	757 760 743 749 753	23 24 23 21 25	0.46 0.48 0.46 0.42 0.50	23 25 23 22 24	0.46 0.50 0.46 0.44 0.48
	12F	738 732 756 737 754	26 23 23 24 22	0.52 0.46 0.46 0.48 0.44	25 23 24 24 21	0.50 0.46 0.48 0.48 0.42
CPA, 40+	11F	758 747 734 748 740	23 24 25 22 25	- - - - -	23 24 26 24 25	0.46 0.48 0.52 0.48 0.50

+ Administered as a single dose

M: Male

F: Female

 

Individual animal data

Data for n-butyl bromide

Treatment (mg/kg/day)	Animal number	PCE:NCE counted	Ratio PCE/NCE	Total PCE Count	MN PCE	PCE/ 1000
Vehicle	716 M 730 M 708 M 707 M 704 M	501 : 535 531 : 639 440 : 773 576 : 478 757 : 470	0.94 0.83 0.57 1.21 1.61	2000 2000 2000 2000 2000	1 0 2 0 0	0.50 0.00 1.00 0.00 0.00
156.3	717 M 711 M 729 M 710 M 713 M	505 : 505 526 : 522 460 : 635 598 : 704 605 : 448	1.00 1.01 0.72 0.85 1.35	2000 2000 2000 2000 2000	1 1 0 2 0	0.50 0.50 0.00 1.00 0.00
312.5	706 M 720 M 714 M 702 M 721 M	633 : 561 694 : 573 447 : 663 721 : 344 540 : 535	1.13 1.21 0.67 2.10 1.01	2000 2000 2000 2000 2000	1 0 1 0 0	0.50 0.00 0.50 0.00 0.00
625	719 M 722 M 705 M 718 M 703 M	540 : 545 718 : 400 529 : 700 342 : 658 422 : 613	0.99 1.08 0.76 0.52 0.69	2000 2000 2000 2000 2000	1 5 1 2 0	0.50 2.50 0.50 1.00 0.00
CPA, 40+	723 M 727 M 701 M 726 M 724 M	524 : 517 665 : 406 604 : 452 668 : 341 570 : 467	1.01 1.64 1.34 1.96 1.22	2000 2000 2000 2000 2000	14 21 15 22 20	7.00 10.50 7.50 11.00 10.00
Vehicle	755 F 745 F 744 F 739 F 742 F	395 : 634 596 : 444 541 : 479 763 : 252 677 : 494	0.62 1.34 1.13 3.03 1.37	2000 2000 2000 2000 2000	2 0 3 4 0	1.00 0.00 1.50 2.00 0.00
312.5	750 F 752 F 759 F 751 F 746 F	797 : 40.1 414 : 665 457 : 701 578 : 426 513 : 498	1.99 0.62 0.65 1.36 1.03	2000 2000 2000 2000 2000	1 0 0 0 0	0.50 0.00 0.00 0.00 0.00
625	736 F 733 F 735 F 741 F 731 F	686 : 634 495 : 611 565 : 561 531 : 662 544 : 494	1.08 0.81 1.01 0.80 1.10	2000 2000 2000 2000 2000	2 1 1 0 0	1.00 0.50 0.50 0.00 0.00
1250	757 F 760 F 743 F 749 F 753 F	570 : 430 539 : 484 372 : 716 551 : 504 546 : 458	1.33 1.11 0.52 1.09 1.19	2000 2000 2000 2000 2000	0 1 2 0 1	0.00 0.50 1.00 0.00 0.50
CPA, 40+	758 F 747 F 734 F 748 F 740 F	545 : 552 494 : 663 614 : 386 549 : 541 633 : 408	0.99 0.75 1.59 1.01 1.55	2000 2000 2000 2000 2000	21 14 15 15 19	10.50 7.00 7.50 7.50 9.50

+ Administered as a single dose

 

Statistical analysis of data

Data for n-butyl bromide

Males:

Treatment (mg/kg/day)	PCE scored	MN PCE observed	MN PCE/ 1000 cells	Hetero-genicity ꭓ2	Signif-icance	2 x 2 Contingency ꭓ2	Signif-icance
Vehicle 156.13 312.5 625 CPA, 40+	10000 10000 10000 10000 10000	3 4 2 9 92	0.30 0.40 0.20 0.90 9.20	5.33 3.50 3.00 8.23	NS NS NS NS	0.00 0.00 2.08 81.90	NS NS NS p≤0.001

Linear trend: z = 1.992, p≤0.05 (Considered to be of negligible importance because there were no significant differences between control and treated groups).

Females:

Treatment (mg/kg/day)	PCE scored	MN PCE observed	MN PCE/ 1000 cells	Hetero-genicity ꭓ2	Signif-icance	2 x 2 Contingency ꭓ2	Signif-icance
Vehicle 312.5 625 1250 CPA, 40+	10000 10000 10000 10000 10000	9 1 4 4 84	0.90 0.10 0.40 0.40 8.40	7.12 4.00 3.50 3.50	NS NS NS NS	4.90 1.23 1.23 59.16	p≤0.05* NS NS p≤0.001

Linear trend: z = 1.036, NS

NS = Not significant

MN = Micronucleated

+ Administered as a single dose

*Represents a statistically significant decrease and therefore of no biological relevance.

 

Historical vehicle control data

Sex		Group Mean Ratio PCE/NCE*	Group mean frequency of micronucleated PCE (per 1000)*	Animal (%) with 0.1 (or more) micronuclei (per 2000 PCE scored)**
				0	1	2	3	4	5(+)
Males	Mean Range	1.09 0.65 – 1.78	0.53 0 – 1.2	36	35	20	6	2	1
Females	Mean Range	1.07 0.67 – 1.62	0.48 0 – 1.1	38	34	19	7	1	1

 *Average of group means from 58 consecutive studies at September 1997

 Data from 24 and 48 hour sampling times are combined

**Individual animal profile based on the above 58 experiments; data from 640 males and 537 females

Applicant's summary and conclusion

Conclusions:

It is concluded that n-butyl bromide did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 625 mg/kg/day (males) or 1250 mg/kg/day (females), doses at which signs of clinical toxicity were observed.

Executive summary:

n-Butyl bromide was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels. The choice of dose levels was based on an initial toxicity range-finding study in which n-butyl bromide, made up in com oil was administered to mice intraperitoneally.

The test article was administered once daily on two consecutive days to groups of mice at doses covering the range 500 to 2000 mg/kg/day. Observations were made over a 4 day period following the second administration and signs of toxicity recorded. Male mice proved to be more sensitive to the test article than females and for the micronucleus test, n-butyl bromide was made up as described and administered at 156.3, 312.5 and 625 mg/kg/day to groups of five male mice and at 312.5, 625 and 1250 mg/kg/day to groups of five female mice. Mice were killed 24 hours after the second administration.

Signs of clinical toxicity were seen among animals of both sex indicating that it would not have been possible to administer the test article at an appreciably higher dose.

 

The negative (vehicle) control in the study was com oil also administered intraperitoneally once daily on two consecutive days. Groups of five male and five female mice treated with this were killed and sampled 24 hours after the second administration. Cyclophosphamide (CPA), the positive control, was dissolved in saline and administered intraperitoneally as a single dose at 40 mg/kg to groups of five male and five female mice which were killed after 24 hours. All positive control animals exhibited increased numbers of micronucleated polychromatic erythrocytes (PCE) such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls (2 x 2 contingency X2 test).

 

Slides from all dose groups were analysed. Negative (vehicle) control mice exhibited normal group mean ratios of PCE to NCE (normochromatic erythrocytes) and normal frequencies of micronucleated PCE within historical negative control ranges. Mice treated with n-butyl bromide at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucleated PCE which were similar to the values for vehicle control groups. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test article.

 

It is concluded that n-butyl bromide did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 625 mg/kg/day (males) or 1250 mg/kg/day (females), doses at which signs of clinical toxicity were observed.