1-bromobutane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro-Ames assay

Negative with and without metabolic activation in Salmonella typhimurium tester strains: TA1535, TA1537, TA98 and TA100 and E.coli strain WP2uvrA.

Genetic toxicity in vitro- - Mouse Lymphoma Assay

Read across to 1-bromopropane. The test substance did not induce any significant increase in the mutation frequency in the first experiment. In the second one, it induced a significant increase in the mutation frequency together with an increase in the number of small colonies at 1500 and 2000 µg/mL.  Under the experimental conditions, the test substance, 1-bromopropane showed mutagenic activity in the mouse lymphoma assay, especially without S9 activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May 2002 and 31ay 2002

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

N/A

Additional strain / cell type characteristics:

other: See any other information on materials and methods section

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the livers of Aroclor 1254-induced Sprague Dawley rats

Test concentrations with justification for top dose:

Six concentrations: 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Dimethyl sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See any other information on materials and methods.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (top application)
Preincubation period: 37oc for ~10hrs.
Analysis for concentration, homogeneity and stability of the test material- not determined.
Vehicle controls were used as negative control.
Vehicle and positive controls were used in parallel with the test material with and without S9 (microsomal enzyme fraction)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF THE TEST MATERIAL TOTOXICITY :
5 doses: 50, 150, 1500 and 5000 µg/plate and a vehicle control were tested.

NUMBER OF REPLICATIONS: 2
Incubation period: 37oc for ~48hr.
- Method: bacterial lawn
Frequency of revertant colonies assessed by Domino colony counter.

Evaluation criteria:

Evaluation of Toxicity:
The compound induced toxicity was evaluated on every plate at the end of the incubation period by studying the aspect of the bacterial lawn and was examined by the mean number of revertant colonies for the toxicity assay.

A compound is considered genotoxic if:
• the number of His+ revertant colonies/plate at one concetration is at least twice the number of spontaneous revertants.
• the increase in the number of revertants is concentration related .

Statistics:

Methods recommended by the UKEMS (5) and normally Dunnett's method of linear regression were used to evaluation of the results.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

seen at 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the preliminary study, the test material exhibited toxicity at 5000 ug/plate to the strains of bacteria (TA100 and EP2uvrA) and this was therefore
maintained as the highest concentration for the definitive study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effect (sparsity of the background lawn with or without a decrease in the number of reverant colonies/plate) was noted at 5000 µg/plate.

ADDITIONAL INFORMATION ON GENOTOXICITY:
No genetic toxicity was induced at any concentration with any of the tester strains. Genotoxocity was shown with the positive control substances
showing the validity of the test system. A second study was conducted with the same concentrations and same tester strains as the first and
produced results in agreement with the original definitive study.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The genotoxic potential of butyl bromide was assessed by the Ames test on four Salmonella typhimurium tester strains: TA1537, TA1535, TA98 and TA100 and 1 Escherichia coli strain WP2uvrA, both in the absence and presence of metabolic activation.
butyl bromide was tested at concentrations ranging from 15 to 5,000 µg/plate.
In all studies, whether in the presence or in the absence of metabolic activation, no increase was observed in the number of His+ revertant colonies/plate at any of the concentrations tested, on the five tester strains, with and without S9 mix.
In conclusion, butyl bromide was not genotoxic in the Ames test, with or without metabolic activation.

Executive summary:

The genotoxic potential of butyl bromide was assessed by the Ames test on five Salmonella typhimurium tester strains: TA1535, TA1537, TA98 and TA100 and E.coli strain WP2uvrA, both in the absence and presence of metabolic activation.

Butyl bromide was tested at concentrations ranging from 15 to 5,000 µg/plate.

No significant increase in the frequency of revertant colonies of bacteria were recorded for any of the strains of Salmonella and E.coli used, at any dose level with or without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13th September - 31st October 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: E.E.C. Directive No. 87/302/E.E.C. 18th November 1987

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: U.S.A./E.P.A./T.S.C.A. Federal Register, Vol. 50, No. 188, Subpart F, 27th September 1985.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK (thymidine kinase) locus in L5178Y mouse lympoma cells. Mutant form is TK-/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y cells are sensitive to chemical mutagens, have a high cloning efficiency and a stable spontaneous mutant frequency.The average cell cycle time is 12-14 hours and the TK phenotypic expression time is two days. Regular checks demonstrated the absence of mycoplasma contamination.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from livers of Aroclor 1254-induced rats

Test concentrations with justification for top dose:

Without activation
First experiment: 125, 250, 500, 1000 and 1500 µg/mL
Second Experiment: 250, 500, 1000, 1250 and 1500 µg/mL

With activation
First Experiment: 125, 250, 500, 1000, 1500 and 2000 µg/mL
Second Experiment: 500, 1000, 1500, 2000 and 2500 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

for tests without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

for tests with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours at 37 ºC
- Exposure duration: 3 hours at 37 ºC
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 ± 1 days
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): Not used

NUMBER OF REPLICATIONS: 2 cultures per dose level.

NUMBER OF CELLS EVALUATED: 2000 cells per well (two 96-well plates/culture = four plates per dose level)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A reproducible two-fold increase in the mutant frequency when compared with the vehicle controls, at any dose-level and/or evidence a dose-relationship were considered as a positive result. Reference to historical data, or other considerations of biological relevance might be also taken into account in the evalulation of the data obtaibed.

Statistics:

Data from the cytotoxicity plates (empty wells) are used to calculate the cloning efficiency after treatment (CE0) and the survival relative to the vehicle controls after treatment (RCE0). These values give a measure of the toxicity of the treatment with the test substance. CE0 is calculated from the zero term of the Poisson distribution.

Data from the viability plates (empty wells) are used to calculate the cloning efficiency at the end of the expression period (CE2) and the viability relative to the vehicle controls at the end of the expression period (RCE2). These values indicate the viability of the cell populations at the end of the expression period. CE2 is calculated from the zero term of the Poisson distribution.

Data from the mutant plates (empty wells) are used to calculate the cloning efficiency after treatment (CE mutant). This value is an indicator of absolute mutant frequency. CE mutant is calculated from the zero term of the Poisson distribution (only mutant clones are able to grow in TFT containing medium).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

seen at 2500 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:

The test substance was freely soluble in the vehicle at 500 µg/mL. Thereafter, a moderate to strong emulsion was observed in the culture medium at the final dose levels of 2500 and 5000 µg/mL. Consequently the maximum dose level was 2500 µg/mL.


ADDITIONAL INFORMATION ON CYTOTOXICITY:

Marked cytotoxicty was recorded at 2500 µg/mL. All the cells were dead after three hours of treatment both with and without S9 activation. At the lower dose levels, a moderate reduction of the cloning efficiency of treated cultures when compared to control cultures was observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Without S9 activation

At 1500 µg/mL, 21 and 33 % RCE₀ were noted when compared to control cultures for the first and second experiments respectively.

At 1250 µg/mL (second experiment), RCE₀ was 46 %.

At lower dose-levels, no significant differences between treated and control were noted.

The number of viable cells two days after treatment (assessed by the cloning frequencies CE₂) was considered equivalent to that of the control cultures.

The test substance induced a reproducible and significant increase in the mutation frequency together with an increase in the number of small colonies at dose levels between 1000 and 1500 µg/mL.

With S9 activation

No significant cytotoxicity was observed in the first experiment, only a moderate reduction of the RCE₀ (59 %) was noted at 2000 µg/mL. In the second experiment, cytotoxicity was more marked:

At 2500 µg/mL, all the cells were dead three hours after treatment

At 2000 µg/mL, RCE₀ was 9 %.

At 1500 µg/mL, RCE₀ was 36 %.

At lower dose-levels, no marked differences between treated and control were noted.

The number of viable cells two days after treatment (assessed by the cloning efficiencies CE₂) was considered to that of the control cultures.

The test substance did not induce any significant increase in the mutation frequency in the first experiment. In the second one, it induced a significant increase in the mutation frequency together with an increase in the number of small colonies at 1500 and 2000 µg/mL.

Conclusions:

Interpretation of results (migrated information):
positive

Under the experimental conditions, the test substance, 1-bromopropane showed mutagenic activity in the mouse lymphoma assay, especially without S9 activation.

Executive summary:

In an in vitro mutagenicity study, the potential of 1 -bromopropane to induce mutations at the TK (thymadine kinase) locus in L5179Y mouse lymphoma cells was examined, according to the OECD 476 guideline. The study was perfomed to GLP with and without metabolic activation using test concentrations from 125 - 2500 µg/mL test substance.

Under the experimental conditions, the test substance, 1-bromopropane showed mutagenic activity in the mouse lymphoma assay, especially without S9 activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across to structural analogue.

Justification for type of information:

Please see attached justification for read across in Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

seen at 2500 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo-mouse bone marrow micronucleus test

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.

Link to relevant study records

Reference

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

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

Specific details on test material used for the study:

No further details specified in the study report.

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.

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.

Dose / conc.:

156.3 mg/kg bw/day

Dose / conc.:

312.5 mg/kg bw/day

Dose / conc.:

625 mg/kg bw/day

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.

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.

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Genetic toxicity in vitro:

The genotoxic potential of butyl bromide was assessed by the Ames test on fiveSalmonella typhimurium tester strains: TA1535, TA1537, TA98 and TA100 and E.coli strain WP2uvrA, both in the absence and presence of metabolic activation.

Butyl bromide was tested at concentrations ranging from 15 to 5,000 µg/plate.

No significant increase in the frequency of revertant colonies of bacteria were recorded for any of the strains of Salmonella and E.coli used, at any dose level with or without metabolic activation.

In an in vitro mutagenicity study, the potential of 1 -bromopropane to induce mutations at the TK (thymadine kinase) locus in L5179Y mouse lymphoma cells was examined, according to the OECD 476 guideline. The study was perfomed to GLP with and without metabolic activation using test concentrations from 125 - 2500 µg/mL test substance.

Under the experimental conditions, the test substance, 1-bromopropane showed mutagenic activity in the mouse lymphoma assay, especially without S9 activation.

Genetic toxicity in vivo:

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.

Justification for classification or non-classification

The positive mouse lymphoma assay result is mitigated by the negative in vivo mouse micronucleus result. The substance is not considered to be classified for mutagenicity on this basis.