1-bromobutane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 January 1996 to 12 February 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

O.E.C.D. guideline No. 406, 17th July 1992

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

E.C. Directive No. 92/69/E.E.C., B6, 31st July 1992.

Deviations:

not specified

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Available study data is over 12 years old.

Test material

Test material information

Constituent 1

Reference substance name:

1-bromobutane

EC Number:

203-691-9

EC Name:

1-bromobutane

Cas Number:

109-65-9

Molecular formula:

C4H9Br

IUPAC Name:

1-bromobutane

Test material form:

liquid

Details on test material:

name:
protocol and labelling: n-BUTYL BROMIDE
batch number:
protocol and labelling: 5-241-1 / 0-201-1
Elf Atochem filing number: CAL 6777/95
description: colourless liquid
quantity and container: 150 gin one glass flask
date of receipt: 19.12.95
storage conditions: at room temperature and protected from light
purity: 99.8%.

Specific details on test material used for the study:

No further details specified in the study report.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Species and strain: Dunkin-Hartley guinea-pigs.
Reason for this choice: species recommended by the international regulations for sensitization studies. The strain used has been shown to produce a satisfactory sensitization response using known positive sensitizers.
Breeder: Centre d'Elevage Lebeau, 78950 Gambais, France.
Number: 30 animals (15 males and 15 nulliparous and non-pregnant females).
Allocation of the animals to the groups: on day -1, the animals were weighed and randomly allocated to two groups: a control group 1 consisting of ten animals (five males and five females) and a treated group 2 consisting of 20 animals (ten males and ten females).
Weight: on day 1, the animals were approximately three months old and had a mean body weight ± standard deviation of 422 ± 20 g for the males and 407 ± 22 g for the females.
Acclimatization: at least five days before the beginning of the study.
Identification of the animals: ear-tattoo.

Environmental conditions
During the acclimatization period and throughout the study, the conditions in the animal room were set as follows:
Temperature: 21 ± 2 °C
Relative humidity: 30 to 70%
Light/dark cycle: 12 h/12 h
Ventilation: about 12 cycles/hour of filtered, non-recycled air.
The temperature and relative humidity were recorded continuously and records retained.
The housing conditions (temperature, relative humidity and ventilation) were checked monthly.
During the acclimatization period and throughout the study, the animals were housed individually in polycarbonate cages (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle.
Dust-free sawdust was provided as litter (SICSA, 92142 Alfortville, France).
Bacteriological analysis of the sawdust and detection of possible contaminants (pesticides, heavy metals) are performed periodically.

Food and water
During the study, the animals had free access to "106 diet" (U.A.R., 91360 Villemoisson-surOrge, France).
Each batch of food was analysed (composition and contaminants) by the supplier.
Drinking water filtered by a F.G. Millipore membrane (0.22 micron) was provided ad libitum.
Bacteriological and chemical analysis of the water and diet and detection of possible contaminants (pesticides, heavy metals and nitrosamines) are performed periodically.
It was verified that no contaminants in the diet or water at levels likely to influence the outcome of the study were present.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

paraffin oil

Concentration / amount:

25% (w/w)

Day(s)/duration:

Day 1/7 days

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

Day 8/48 hours

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

50% (w/w) - left flank

Day(s)/duration:

Day 22/48 hours

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

25% (w/w) - right flank

Day(s)/duration:

Day 22/48 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Thirty guinea-pigs were allocated to two groups: a control group 1 (five males and five females) and a treated group 2 (ten males and ten females).

Details on study design:

Preliminary test
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.

By intradermal route:
24 hours before treatment, the dorsal region of the animals was clipped, the test substance was prepared in an appropriate vehicle, intradermal administrations of the test substance (0.1 ml) at different concentrations were performed in the dorsal region between the shoulders, cutaneous reactions were evaluated approximately 24, 48 hours and six days after injection.

By cutaneous route:
24 hours before treatment, both flank regions of the animals were clipped, if necessary, the test substance was prepared in an appropriate vehicle, the test substance (0.5 ml for each concentration) was applied to a dry gauze pad of approximately 4 cm2 which was held in place by an occlusive dressing for 24 hours, cutaneous reactions were evaluated approximately 24 and 48 hours after removal of the dressings.

Criteria for selection of concentrations
The following criteria were used:
-the concentrations should be well-tolerated systemically and locally,
-intradermal injections should cause moderate irritant effect (no necrosis or ulceration of the skin),
-topical application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration,
-topical application for the challenge should be the highest concentration which does not cause irritant effect.

Main study
Preparation of the animals
For all animals and before each treatment, the application sites were:
-clipped on days -l and 7 (scapular area 4 cm x 2 cm),
-clipped and shaved on day 21 (each flank 2 cm x 2 cm).

Induction phase by intradermal and cutaneous routes
Intradermal route
On day 1, six injections were made deep into the dermis of a clipped area (4 cm x 2 cm) in the dorsal region between the shoulders, using a needle (diameter: 0.50 x 16 mm, Terumo: C.M.L., 77140 Nemours, France) mounted on a 1 ml glass syringe (0.01 ml graduations, Record: Carrieri, 75005 Paris, France).

Cutaneous route
On day 7, the scapular area was clipped.
As the test substance was shown to be irritant during the preliminary tests, a topical application with sodium laurylsulphate was not necessary on day 7.
On day 8, a topical application to the region of the intradermal injections (4 cm x 2 cm) was performed.

Control group
-application of 0.5 ml of the vehicle.

Treated group
-application of 0.5 ml of the test substance at the chosen concentration.

The test substance and the vehicle were prepared on a dry gauze pad (Semes France, 54183 Heillecourt, France), which was then applied to the dorsal region between the shoulders and held in place for 48 hours by means of an adhesive hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Medicales, 92240 Malakoff, France).
On removal of the dressing, if present, any residual test substance was removed by means of a dry or a moistened gauze pad.
Cutaneous reactions were recorded one hour after removal of the occlusive dressing.

Challenge phase
On day 22, the animals from both groups received an application of 0.5 ml of the test substance at a concentration of 25% (w/w) to the posterior right flank, and 0.5 ml of the test substance at a concentration of 50% (w/w) to the posterior left flank. This application was performed using a 1 ml plastic syringe (0.01 ml graduations, Terumo: C.M.L., 77140 Nemours, France). The test substance and vehicle were prepared on a dry gauze pad (Semes France, 54183 Heillecourt, France), then applied to a 4 cm2 (2 cm x 2 cm) clipped area of the skin. The gauze pad was held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Medicales, 92240 Malakoff, France).
On removal of the dressing, if present, any residual test substance was removed by means of a dry or a moistened gauze pad.

CLINICAL EXAMINATIONS
The animals were observed twice a day during the study in order to check for clinical signs and mortality.

BODY WEIGHT
The animals were weighed individually on the day of allocation into the groups, on the first day of the study (day 1), on days 8 and 15 and on the last day of the study.

PATHOLOGY
Necropsy
Macroscopic examination of the main organs was performed on the animal found dead during the study.
At the end of the study, all the surviving animals were killed by CO, inhalation in excess. No necropsy was performed.

Cutaneous samples
No skin samples were taken.

Microscopic examination
No histological examinations were performed.

Positive control substance(s):

yes

Remarks:

2,4-dinitro chlorobenzene.

Results and discussion

Positive control results:

The guinea-pigs which were used in a recent study, showed a satisfactory sensitization response in 100% animals using a positive sensitizer.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50% (w/w) - left flank

No. with + reactions:

0

Total no. in group:

10

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25% (w/w) - right flank

No. with + reactions:

0

Total no. in group:

10

Results 3

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50% (w/w) - left flank

No. with + reactions:

0

Total no. in group:

10

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25% (w/w) - right flank

No. with + reactions:

0

Total no. in group:

10

Results 5

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50% (w/w) - left flank

No. with + reactions:

3

Total no. in group:

19

Clinical observations:

At the 24-hour reading, very slight erythema (grade 1) was observed in 3/19 and 2/19 animals treated with 50% and 25% (w/w) of the test substance, respectively.

Remarks on result:

other: Since skin irritation was noted in the preliminary assay, with the undiluted test substance, these very faint and transient skin reactions were attributed to irritant properties of the test substance.

Results 6

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25% (w/w) - right flank

No. with + reactions:

2

Total no. in group:

19

Clinical observations:

At the 24-hour reading, very slight erythema (grade 1) was observed in 3/19 and 2/19 animals treated with 50% and 25% (w/w) of the test substance, respectively.

Remarks on result:

other: Since skin irritation was noted in the preliminary assay, with the undiluted test substance, these very faint and transient skin reactions were attributed to irritant properties of the test substance.

Results 7

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50% (w/w) - left flank

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

No skin reactions were observed at the 48-hour reading.

Remarks on result:

no indication of skin sensitisation

Results 8

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25% (w/w) - right flank

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

No skin reactions were observed at the 48-hour reading.

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

PRELIMINARY STUDY

Administration by intradermal route

Several test were performed in order to determine the concentration to be used in the main study.

Animal number	Concentration of the test substance % (w/w)	Scoring after treatment
		24 hours	48 hours	6 days
Male 01	10 25 50	Irritation Irritation Necrosis	Irritation Irritation Necrosis	Irritation Irritation Crusts
Female 01	10 25 50	Irritation Irritation Necrosis	Irritation Irritation Necrosis	Irritation Irritation Crusts

Concentration chosen for the main study was 25% (w/w).

 

Application by cutaneous route

One test was performed in order to check if the undiluted compound was irritant.

Animal number	Concentration of the test substance %		Scoring after removal of the dressing^(1)
			24 hours		48 hours
			E	O	E	O
Male 01	100 100	RF LF	1 1	0 0	1 1	0 0
Female 01	100 100	RF LF	1 1	0 0	1 1	0 0
Male 02	50 (w/w) 50 (w/w)	RF LF	0 0	0 0	0 0	0 0
Female 03	50 (w/w) 50 (w/w)	RF LF	0 0	0 0	0 0	0 0

E: Erythema

O: Oedema

RF: Right flank

LF: left flank

(1): No residual test substance was observed.

 

Concentration chosen for the topical application of the induction phase (day 8) was 100%.

For the challenge application, it was 25% (w/w) for the right flank and 50% (w/w) for the left flank.

 

MAIN STUDY

Clinical examinations

No clinical signs were observed during the study.

One male of the treated group (No. 14) was found dead on day 18. Spontaneous mortality are often observed in guinea-pigs and this mortality was not attributed to treatment.

The body weight gain of the treated animals was normal when compared to that of the control animals.

 

Scoring of cutaneous reactions

End of the induction period

On day 10, after topical application of the induction period, signs of irritation were observed at the test site (dorsal region between shoulders) in the control and treated groups.

 

Challenge application

NO residual test substance was observed after removal of the dressing.

Skin reactions were as follows:

Sex	Animal number	Control group
		24 hours				48 hours
		Erythema		Oedema		Erythema		Oedema
		LF	RF	LF	RF	LF	RF	LF	RF
Male	01 02 03 04 05	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0
Female	16 17 18 19 20	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0

LF: left flank (test substance at a concentration of 50% (w/w))

RF: right flank (test substance at a concentration of 25% (w/w))

 

Sex	Animal number	Treated group
		24 hours				48 hours
		Erythema		Oedema		Erythema		Oedema
		LF	RF	LF	RF	LF	RF	LF	RF
Male	06 07 08 09 10 11 12 13 14 15	0 0 1 0 0 1 0 0 - 1	0 0 1 0 0 0 0 0 - 1	0 0 0 0 0 0 0 0 - 0	0 0 0 0 0 0 0 0 - 0	0 0 0 0 0 0 0 0 - 0	0 0 0 0 0 0 0 0 - 0	0 0 0 0 0 0 0 0 - 0	0 0 0 0 0 0 0 0 - 0
Female	21 22 23 24 25 26 27 28 29 30	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0

LF: left flank (test substance at a concentration of 50% (w/w))

RF: right flank (test substance at a concentration of 25% (w/w))

-: dead animal

 

At the 24-hour reading, very slight erythema (grade 1) was observed in 3/19 and 2/19 animals treated with 50% and 25% (w/w) of the test substance, respectively.

No skin reactions were observed at the 48-hour reading.

 

Since skin irritation was noted in the preliminary assay, with the undiluted test substance, these faint and transient skin reactions were attributed to irritant properties of the test substance.

 

Pathology

Necropsy

Macroscopic examination of the main organs of the animal No. 14, found dead during the study, revealed no apparent abnormalities.

 

Microscopic examination

No microscopic examinations were performed.

 

INDIVIDUAL BODY WEIGHT VALUES

(g)

Groups	Sex	Animals	Days
			-1	1	(1)	8	(1)	15	(1)	25
1	Male	01 02 03 04 05   M SD	432 436 411 400 418   419 15	426 438 414 408 433   424 13	87 86 113 91 52   86 22	513 524 527 499 485   510 18	53 32 11 -6 8   20 23	566 556 538 493 493   529 35	11 -64 -28 43 -4   -8 40	577 492 510 536 489   521 37
	Female	16 17 18 19 20   M SD	394 418 413 395 435   411 17	401 404 413 389 438   409 18	109 83 70 92 86   88 14	510 487 483 481 524   497 19	-14 -26 21 -42 6   -11 25	496 461 504 439 530   486 36	6 72 0 54 -8   25 36	502 533 504 493 522   511 16
2	Male	06 07 08 09 10 11 12 13 14 15   M SD	418 448 438 397 429 458 409 384 431 385   420 26	420 453 434 403 422 453 408 385 435 399   421 23	89 53 65 52 30 84 87 53 66 72   65 19	509 506 500 455 452 537 495 438 501 471   486 31	77 4 51 45 22 35 -20 40 -96 -14   14 49	586 510 551 500 474 572 475 478 405 457   501 56	-58 41 -21 -3 64 -14 101 26   71   23 51	528 551 530 497 538 558 576 504   528   534 25
	Female	21 22 23 24 25 26 27 28 29 30   M SD	395 400 394 442 383 412 432 403 420 361   404 24	399 409 396 443 383 426 418 396 428 362   406 24	66 73 84 43 70 70 43 81 86 62   68 15	465 482 480 486 453 496 461 477 514 424   474 25	0 1 14 36 11 -2 -11 33 36 31   15 18	465 483 494 522 464 494 450 510 550 455   489 32	22 41 16 -3 68 -5 57 22 -31 -7   18 31	487 524 510 519 532 489 507 532 519 448   507 26

(1) = body weight gain

M = Mean

SD = Standard Deviation

Animal found dead during the study not mentioned

 

Positive control to check the sensitivity of Dunkin-Hartley guinea-pigs

Test substance: 2,4-dinitro chlorobenzene

Number of animals: ten males and ten females

Induction:      0.1% intradermal route day 1

                       1% cutaneous route day 8

Challenge application:1% right flank

                                   Paraffin oil left flank

 

Conclusion

Under the experimental conditions and according to the Magnusson and Kligman method, 2,4-dinitro chlorobenzene at a concentration of 1% (w/w) induced positive skin sensitization reactions in 100% of the guinea-pigs.

 

INDIVIDUAL REACTIONS: CHALLENGE PHASE

MACROSCOPIC FINDINGS

Group

Sex

Animals

24-hour

48-hour

Erythema

Oedema

Erythema

Oedema

Conclusion

LF

RF

LF

RF

LF

RF

LF

RF

LF

RF

Treated

Male

81 82 83 84 85 86 87 88 89 90

0 0 0 0 0 0 0 0 0 0

1/C 2/S 1 2 2/C/S 1 2/A 2 1 2

0 0 0 0 0 0 0 0 0 0

0 0 0 2 0 0 2 0 0 4

0 0 0 0 0 0 0 0 0 0

0/C/S 1/C/S 0/C/S 0/C/S 2/C/A 1/C/S 4/A/C 0/C/S 0/S 2/C/S

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

- - - - - - - - - -

+ + + + + + + + + +

Female

96 97 98 99 100 101 102 103 104 105

0 0 0 0 0 0 0 0 0 0

2 1 1/C 2 2 2/A 2/S 2 3 1

0 0 0 0 0 0 0 0 0 0

2 0 0 0 2 0 0 0 2 0

0 0 0 0 0 0 0 0 0 0

2/C/S 0/S 0/C/S LS 0/C/S LS/A 2/C/S 2/C/S 4/S LS

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 2 0

- - - - - - - - - -

+ + + + + + + + + +

-: negative

+: hypersensitizing reactions

C: yellow colouration of the skin due to the test substance

S: dryness of the skin

A: crusts

LS: scoring masked by a marked dryness of the skin

LF: left flank

RF: right flank

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions and according to the maximization method of Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of the test substance n-BUTYL BROMIDE were observed in guinea-pigs.

Executive summary:

At the request of Elf Atochem S.A., Paris-la-Defense, France, the potential of the test substance n-BUTYL BROMIDE to induce delayed contact hypersensitivity was evaluated in guinea-pigs according to the maximization method of Magnusson and Kligman and to O.E.C.D. (No. 406, 17th July 1992) and E.C. (92/69/E.E.C., B6) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

Methods

Thirty guinea-pigs were allocated to two groups: a control group 1 (five males and five females) and a treated group 2 (ten males and ten females).

 

On day 1, in the dorsal region between the shoulders, intradermal injections of Freund's complete adjuvant mixed with the test substance (treated group) or the vehicle (control group) were prepared.

 

On day 8, this same test site was treated by topical application of the test substance (treated group) or the vehicle (control group) and was covered by an occlusive dressing for 48 hours.

 

After a rest period of 12 days, all animals of the treated and control groups were challenged by a topical application of the test substance. The right flank received the test substance at a concentration of 25% (w/w), the left flank received the test substance at a concentration of 50% (w/w).

 

Test substance and vehicle were maintained under an occlusive dressing for 24 hours. Skin reactions were evaluated approximately 24 and 48 hours later.

 

Test substance concentrations were as follows:

Induction (treated group)

-intradermal injections: n-BUTYL BROMIDE at 25% (w/w) in paraffin oil

-topical application: n-BUTYL BROMIDE at 100%.

 

Challenge (all groups)

-topical application: n-BUTYL BROMIDE at 50% (w/w) in paraffin oil on the left flank,

-topical application: n-BUTYL BROMIDE at 25% (w/w) in paraffin oil on the right flank.

 

At the end of the study, animals were killed. No skin samples were taken from the challenge application sites.

 

The sensitivity of the guinea-pigs in C.I.T. experimental conditions were checked in a recent study with a positive sensitizer: 2,4-dinitro chlorobenzene. During induction period, the test substance was applied at 0.1% (day 1) and 1% (day 8) concentrations. At cutaneous challenge application, 1% (w/w) was tested on the right flank.

 

Results

No clinical signs and no deaths related to treatment were noted during the study.

No well-defined cutaneous reactions were observed after the challenge application.

The guinea-pigs which were used in a recent study, showed a satisfactory sensitization response in 100% animals using a positive sensitizer.

 

Conclusion

Under the experimental conditions and according to the maximization method of Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of the test substance n-BUTYL BROMIDE were observed in guinea-pigs.