Bronopol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted according to the acknowledged method of Magnusson B and Kligman AM (J. Invest. Dermatol., 52, 268-276, 1969), which preceeded OECD 406.GLP was not compulsory at the time the study was conducted. Few deficiencies were noticed (no positive control; no specification, whether the application sites needed to be cleaned from test substance residues following removal of the test patch; the control groups (treated and untreated) consisted each of 4 animals instead of five, as recommended by OECD guideline) but they do not affect the validity of the reported data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1976

Materials and methods

Principles of method if other than guideline:

The study was conducted according to the acknowledged method of Magnusson B and Kligman AM (J. Invest. Dermatol., 52, 268-276, 1969), which preceeded OECD 406
The method closely followed that of Magnusson and Kligman (1969), however with following deviation: each test was performed with 10 treated animals, 4 treated controls and 4 untreated controls. Furthermore, the test was conducted with 4 challenges, instead of one as prescribed by Magnusson and Kligman (1969).

GLP compliance:

no

Remarks:

GLP was not compulsory at the time the study was conducted.

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study was performed before LLNA-method was described as OECD test guideline and before it could be established in the EU-REACH regulation.

Test material

Test material information

Constituent 1

Reference substance name:

Bronopol

EC Number:

200-143-0

EC Name:

Bronopol

Cas Number:

52-51-7

Molecular formula:

C3H6BrNO4

IUPAC Name:

2-bromo-2-nitropropane-1,3-diol

Test material form:

not specified

Details on test material:

Purity 100%

Specific details on test material used for the study:

Purity 100%

In vivo test system

Test animals

Species:

guinea pig

Sex:

male/female

Details on test animals and environmental conditions:

Mean weight at test initiation: 320 g.

Study design: in vivo (non-LLNA)

Induction

Route:

other: 2 induction applications.

Vehicle:

other: physiological saline, distilled water

Concentration / amount:

1st application: Induction 0.02 % intracutaneous
2nd application: Induction 1.5 % occlusive epicutaneous
3rd application: Challenge 0.4 % occlusive epicutaneous

Challenge

Route:

epicutaneous, occlusive

Vehicle:

other: physiological saline, distilled water

Concentration / amount:

1st application: Induction 0.02 % intracutaneous
2nd application: Induction 1.5 % occlusive epicutaneous
3rd application: Challenge 0.4 % occlusive epicutaneous

No. of animals per dose:

Main sensitisation test:
treated group: 6 females and 4 males
treated control group: 4 females
untreated control group: 4 females

Details on study design:

The test concentrations selected for induction (intradermal injection and epicutaneous occlusive) and challenge were chosen on the basis of the results of two preliminary tests (intradermal injection and topical application) conducted respectively with 4 female animals. The test concentrations for the main sensitisation test were: 0.02% in 0.9% saline for the first induction (intradermal injection), 1.5% in distilled water for the second induction (epicutaneous, occlusive) and 0.04% in distilled water for challenge. The main test was conducted with 50% Freunds Complete Adjuvant, FCA, in saline and consisted of following groups: a treated group with 10 animals (6 females and 4 males), a treated control group with 4 females (induced as treated animals but without test substance; challenge similar to that of treated animals) and an untreated control group with 4 females (untreated animals challenged exactly as treated animals). First induction: 6 x 0.1 ml injections within a 2 cm x 4 cm area of the shoulder region: 2 injections of test substance in solvent, 2 injections of test substance in 50% CFA in saline and 2 injections of 50% CFA in saline. Second induction: occlusive epicutaneous conditions. Application of a 2 cm x 4 cm patch saturated with test substance over the shoulder injection site, covered with an occlusive dressing.Challenges 1 -3: occlusive epicutaneous conditions as above. The first challenge was conducted after 14 days following the second induction; further challenges (3) were conducted at weekly or greater intervals. Fourth challenge: a fourth challenge was added to (1) to check whether sensitization potential is reduced at lower challenge concentrations (test concentrations: 0.2 and at 0.4% in distilled water) and (2) to look for possible cross-challenge, using a 0.5% solution of 40% formaldehyde. The animals were examined for skin reactions: the application site was examined after 24h and 48h following removal of the patch; the findings were scored 0 to +++ (0 indicates no skin reaction; from + upwards, reactions are considered positive, however no reactions must be seen in controls). Furthermore, the body weights of the animals were recorded weekly.

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.4%

No. with + reactions:

0

Total no. in group:

10

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.4%

No. with + reactions:

0

Total no. in group:

10

Results 3

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

0.4%

No. with + reactions:

0

Total no. in group:

10

Results 4

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

0.4%

No. with + reactions:

1

Total no. in group:

10

Results 5

Reading:

other: third challenge

Hours after challenge:

24

Group:

test chemical

Dose level:

0.4%

No. with + reactions:

2

Total no. in group:

10

Results 6

Reading:

other: third challenge

Hours after challenge:

48

Group:

test chemical

Dose level:

0.4%

No. with + reactions:

2

Total no. in group:

10

Any other information on results incl. tables

Within the challenge 4, a cross-challenge was conducted with formaldehyde, as bronopol is reputed to produce formaldehyde under certain conditions. Cross-challenge with formaldehyde (occlusive epicutaneous), 24 h - findings Group Animals Result Evaluation of Sensitization Treated (0.5% of 40% formaldehyde in distilled water) 6 females 4 cases with barely perceptible erythema 0/9 3 males (1 animal killed because of ssevere neck ulceration) 1 case with barely perceptible erythema Cross-challenge with formaldehyde (occlusive epicutaneous), 48 h - findings Group Animals Result Evaluation of Sensitization Treated (0.5% of 40% formaldehyde in distilled water) 6 females No effects 0/9 3 males (1 animal killed because of ssevere neck ulceration) No effects The results indicate that formaldehyde was not responsible for the positive reactions reported for the remaining challenges.

The fourth challenge was conducted to see whether the sensitising potential of bronopol decreases with decreasing test concentration. Challenge 4 (occlusive epicutaneous), 24 h - findings Group Animals Result Evaluation of Sensitization Treated (0.2% bronopol in distilled water) 6 females 1 case with scattered, mild erythema 2 cases with barely perceptible erythema 1/9 3 males (1 animal killed because of severe neck ulceration) 1 case with barely perceptible erythema Treated (0.4% bronopol in distilled water) 6 females 1 case with scattered, mild erythema 3 cases with barely perceptible erythema 0/9 3 males (see above) 1 case with barely perceptible erythema The findings indicate that the sensitisation was not reduced at 0.2% challenge. Challenge 4 (occlusive epicutaneous), 48 h - findings Group Animals Result Evaluation of Sensitization Treated (0.2% bronopol in distilled water) 6 females 2 cases with scattered, mild erythema 1 cases with barely perceptible erythema 2/9 3 males (1 animal killed because of severe neck ulceration) No effects Treated (0.4% bronopol in distilled water) 6 females 1 case with scattered, mild erythema 1 case with barely perceptible erythema 1/9 (?) 3 males (see above) No effects

The main 48h - results for the different challenges can be summarized as follows: Challenge 1 (occlusive epicutaneous), 48 h - findings Group Animals Result Evaluation of Sensitization Treated (0.4% bronopol in distilled water) 6 females 1 case with barely perceptible erythema 0/10 The effects seen in treated animals were not indicative of sensitisation, as similar effects also were seen in untreated controls. 4 males 2 cases with barely perceptible erythema Treated control (0.4% bronopol in distilled water) 4 females No effects Untreated controls 4 females 2 cases with barely perceptible erythema. Challenge 2 (occlusive epicutaneous), 48 h - findings Group Animals Result Evaluation of Sensitization Treated (0.4% bronopol in distilled water) 6 females 1 case with scattered, mild erythema 1 case with barely perceptible erythema 4 cases without effects 1/10 4 males 1 case with barely perceptible erythema 3 cases without effects Untreated controls 4 females No effects Challenge 3 (occlusive epicutaneous), 48 h - findings Group Animals Result Evaluation of Sensitization Treated (0.4% bronopol in distilled water) 6 females 2 cases with moderate and diffuse erythema 1 case with barely perceptible erythema 2/10 4 males No effects Untreated controls 4 females No effects A sensitisation was observed in 1 animal after 48 hours following the second challenge; after 48 hours following the third challenge, 2 animals still were sensitised.

The main 24 h - results for the different challenges can be summarized as follows: A sensitisation was observed in 2 animals after 24 hours following the third challenge. Challenge 1 (occlusive epicutaneous), 24 h - findings Group Animals Result Evaluation of sensitization Treated (0.4% bronopol in distilled water) 6 females 1 case with scattered, mild erythema 1 case with barely perceptible erythema 0/10 The effects seen in treated animals including the one case of scattered mild erythema, were not indicative of sensitisation, as similar effects also were seen in untreated controls. 4 males 3 cases with barely perceptible erythema Treated control (0.4% bronopol in distilled water) 4 females No effects Untreated controls 4 females 2 cases with barely perceptible erythema Challenge 2 (occlusive epicutaneous), 24 h - findings Group Animals Result Evaluation of sensitization Treated (0.4% bronopol in distilled water) 6 females 3 cases with barely perceptible erythema 0/10 The effects seen in treated animals were not indicative of sensitisation, as similar effects also were seen in untreated controls. 4 males 1 case with barely perceptible erythema Untreated controls 4 females 3 cases with barely perceptible erythema Challenge 3 (occlusive epicutaneous), 24 h - findings Group Animals Result Evaluation of sensitization Treated (0.4% bronopol in distilled water) 6 females 2 cases with moderate and diffuse erythema 1 case with scattered mild erythema 1 case with barely perceptible erythema 2/10 4 males 1 case with scattered mild erythema 2 cases with barely perceptible erythema Untreated controls 4 females 1 case with barely perceptible erythema

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met