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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
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. 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.
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
Species:
guinea pig
Sex:
male/female
Details on test animals and environmental conditions:
Mean weight at test initiation: 320 g.
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
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.



Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.4%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.4%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.4%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.4%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
0.4%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 0.4%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
0.4%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 0.4%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
other: third challenge
Hours after challenge:
24
Group:
test group
Dose level:
0.4%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
other: third challenge
Hours after challenge:
48
Group:
test group
Dose level:
0.4%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading:

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

Interpretation of results:
not sensitising
Remarks:
Migrated information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Guinea pig maximisation test (defined exposure scheme)

The sensitising potential of Bronopol (purity 100%) was tested in the guinea pig maximisation test. The study (Unilever Research Laboratory, 1976) followed no guideline as it was conducted in 1976; however the study was conducted according to the acknowledged method of Magnusson and Kligman (1969), which preceeded OECD 406. GLP was not compulsory at the time the study was conducted.

The test concentrations selected for induction (intradermal injection and epicutaneous occlusive application) and challenge were chosen on the basis of the results of two preliminary tests (intradermal injection and topical application) conducted each 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 application) and 0.4% 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: Six 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 at the application site after 24 and 48 hours 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). The body weights of the animals were recorded weekly.

Main results: Challenges 1 –3: a sensitisation was observed in 2 animals after 24 hours following the third challenge.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.

Challenge 4: The findings of the fourth challenge indicated that the sensitisation was not reduced at 0.2% challenge. Moreover, the cross-challenge testing revealed that formaldehyde was not responsible for the positive reactions reported for the remaining challenges.

The test was conducted with 4 challenges, instead of one as prescribed by Magnusson and Kligman (1969). After the first challenge, no signs indicative of sensitisation were seen; in fact, sensitisation was observed after the second and third challenge and the percentage of positive reactions was 20%.

Conclusively, under the test conditions chosen, Bronopol showed no skin sensitising potential. The study is classified as key study. With regard to deviations, each test was performed with 10 treated animals, 4 treated controls and 4 untreated controls.

 

Reference: Magnusson B and Kligman AM (1969). The identification of contact allergens by animal assay. The guinea pig maximisation test. J. Invest. Dermatol. 52: 268-276.

 

 

Human patch tests (undefined pre-exposure)

In a human patch test (Boots Company, 1977), Bronopol at a concentration of 0.25% showed no skin sensitizing activity. The study protocol consisted of a three-week induction period with 5% Bronopol, an irritant concentration, followed by a two-week incubation period, and an elicitation with Bronopol at a sub-irritant concentration (0.25 %).The authors concluded that in previous studies false positive skin sensitizing activity was seen due to the high concentration of Bronopol (2.5%) tested (in subsequent studies this concentration was recognized as irritant per se, and thus unsuitable for use in challenge).

Further patch testing (Bryce, 1978) in 149 patients showed Bronopol to be a mild irritant when applied in petrolatum at 0.25 %, but no evidence of sensitization was noticed in this study. Furthermore, a patch testing with Bronopol performed by Maibach (1977) in 8 and 120 subjects with 0.1, 0.5, 1, 2.5 and 5 % Bronopol in soft paraffin and 5 % (induction) and 0.25 % (challenge) Bronopol in soft paraffin demonstrated that the irritancy threshold was approximately 0.5-1 %. No evidence of sensitization was observed at 0.25 %. Thus, false positive results may be obtained in patch tests by the use of Bronopol concentrations above 0.5 %.

Some reliable human patch test data are also available. These exposure related observations in humans demonstrate that dermal exposure of volunteers to the test substance Bronopol results in signs of irritation and a clear positive skin reaction (Schnuch, 1998). It has to be pointed out that Schnuch (1998) investigated the patch test reaction to Bronopol (0.5 % in petrolatum) in 11443 patients (preservative series) and 1781 patients (industrial biocides). In 134 cases (1.2 %) the patch test reaction to Bronopol (0.5 % in pet.) in preservative series and 32 cases (1.8 %) in industrial series showed positive reactions. Conclusively, sensitization rates seem to be quite low in this population. Investigations of Frosch (1986) demonstrated also a low incidence of positive patch test reactions to Bronopol. Furthermore, Ford (1986) postulated that positive results in patch testing may be seen by approaching the irritancy threshold of Bronopol (0.5 – 1 %).

In general, Bronopol is widely used as a preservative in cosmetics and toiletries. Allergic contact dermatitis to Bronopol has been reported, in a patch test with Bronopol in different concentrations (0.25 %, 0.5 %, 1 % Bronopol), 23 subjects showed irritant reactions to 1 % Bronopol and revealed responses also at lower concentrations (Peters, 1983). Allergic reactions were observed after dermal exposure to Bronopol in patients, too (Shaw, 1997). Bronopol as broad spectrum antimicrobial was assessed using the DRAIZE procedure on normal human test subjects (Marzulli and Maibach, 1973). Human test results showed that at 5 % induction concentration it has a strong skin sensitization potential. Under alkaline conditions, Bronopol liberates formaldehyde. In some subjects who were sensitive to Bronopol, skin reactions were elicited when the subjects were tested with formalin. This suggests that there were other antigenic determinants in the test substance in addition to formaldehyde (Marzulli and Maibach, 1973, Antimicrobials: Experimental contact sensitization in man, J. Soc. Cosmet. Chem. 24, 399-421). Patients with dermatitis showed showed a positive skin reaction to Bronopol at 1% (Rudner, 1997) or at 0.5 % and 0.25 % (Storrs, 1983).

With regard to workers, the skin of 20/50 Bronopol exposed workers showed signs of skin irritation and/or superficial burns following exposure to saturated aqueous solutions or powder of Bronopol on at least one occasion. The reported findings were described as irritant reaction rather than contact allergy (Anonymous, 1984).

 

Corresponding references and endpoint study records are listed in chapter 7.10 (7.10.2; 7.10.3; 7.10.4) and/or 7.12.


Migrated from Short description of key information:
Bronopol is not a skin sensitizer based on a study using guinea pigs and showed no evidence of sensitization in several human patch tests (in congruence with REACH, testing for induction of sensitisation in humans is no longer conducted on ethical grounds). However, further sensisation data on humans are available, these exposure related observations demonstrate that dermal exposure of volunteers to the test substance at non-irritating concentrations may result in signs of irritation and a positive skin reaction. Thus, Bronopol demonstrated in clinical case studies a weak sensitizing potential, independent from release of formaldehyde. Please refer also to section 7.10.4 (epidemiological data).

Justification for selection of skin sensitisation endpoint:
The key study was selected. This 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.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Bronopol is currently not classified as a skin sensitiser according to Directive 67/548/EEC or Regulation (EC) No 1272/2008 and 286/2011.

Human data from clinical epidemiological studies showed ambiguous evidence that bronopol has sensitizing properties. Some studies showed positive reactions in human patch tests, which in some cases might be and in other cases are clearly false-positive, due to irritant properties of bronopol. The overall incidence of positive reactions was very low (approx. 1%) and the degree of severity in the skin reactions was low. In several other studies even no positive reactions were observed. The preexposure towards bronopol of the individuals tested and the purity of bronopol solutions used for patch test analysis often were not documented. Additionally, the concentrations used for induction and/or elicitation were often very high, i.e. irritating, thus the difference between irritant and sensitizing reactions were difficult to distinguish.

Furthermore, the REACh guidance considers data from animal experiments as more reliable arguments that human clinical data (Regulation 286/2011; 3.4.2.2.4.2.: "Evidence from animal studies is usually much more reliable than evidence from human exposure.However, in cases where evidence is available from both sources, and there is conflict between theresults, the quality and reliability of the evidence from both sources must be assessed in order to resolve the question of classification on a case-by-case basis. Normally, human data are not generated in controlled experiments with volunteers for the purpose of hazard classification but rather aspart of risk assessment to confirm lack of effects seen in animal tests. Consequently, positive humandata on skin sensitisation are usually derived from case-control or other, less defined studies. Evaluation of human data must therefore be carried out with caution as the frequency of cases reflect, inaddition to the inherent properties of the substances, factors such as the exposure situation, bioavailability, individual predisposition and preventive measures taken. [...]"), because the documentation and the testing is much more defined. In OECD guideline-conform standard animal studies, bronopol has no sensitizing properties.

Therefore, in a weight of evidence approach we conclude that bronopol might have some weak sensitizing properties, but that this evidence is not enough for classification according to the above mentioned directive or regulations. In addition, due to the irritant properties of bronopol, exposure is sufficiently excluded by risk management measures in the respective uses and therefore there is no risk of potential sensitization.