Registration Dossier

Administrative data

Description of key information

Bronopol is an irritant to skin and mucous membranes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP. However, no data on test substance purity were given.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Principles of method if other than guideline:
USA EPA FIFRA (40 CFR 158, 81-5)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Source: David Percival Ltd., Moston, Sandbach, Cheshire, UK
Weight at study initiation: 2,35 - 3,07 kg
Age: 12 to 16 weeks old.
Sex not specified.
Type of coverage:
semiocclusive
Preparation of test site:
other: clippping of the fur
Vehicle:
water
Controls:
no
Amount / concentration applied:
Concentration: 0.5 g (moistened with 0.5 ml distilled water)
Duration of treatment / exposure:
4 hours
Observation period:
14 days
Number of animals:
6
Details on study design:
The test group consisted of 6 New Zealand White rabbits. About 24 hours prior testing, the fur of the dorsal and flank area of each rabbit was clipped. The test material was prepared by moistening 0.5 g of test substance with 0.5 ml of distilled waterand was applied on a selected shorn skin area, on the back of each rabbit. The application site was covered by means of a patch, which again was maintained in place using a strip of adhesive tape. Furthermore, the trunk of each rabbit was wrapped in a corset to prevent the animals from interfering with the patches. Exposure period was 4 hours; thereafter removal of the dressing and the patch, residual test material on the application site of each rabbit was gently removed using cotton woolsoaked in distilled water. The scoring of the dermal findings was performed according to Draize JH (The appraisal of the safety of chemicals in foods, drugs and cosmetics. Association of Food and Drug Officials of the United States, Austin, Texas, 1959); the reading time points were 60 min, 24 h, 48 h and 72 h after removal of the patches. Additional readings were done on day 7 and day 14.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
other: 24-72h
Score:
6.2
Reversibility:
not fully reversible within: 72 h
Irritant / corrosive response data:
One hour after removal of the dressing: all animals displayed very slight or moderate to severe erythema on the application sites (graded 1 to 3). Slight haemorrhage of the dermal capillaries, brown discoloration and small areas of blanching were seen, which resulted in an average score over all sixanimals of 2.5.
After 24 hours: erythema became severe (graded 4) in 4/6 cases and was accompanied by green/brown coloured necrosis over the whole application site. Small areas of blanching were seen in 5/6 animals, and slight hemorrhage of the dermal capillaries was seen in two of them. In two rabbits, the reaction area was extended to 3 to 4 cm beyond the application site. The average score over all six animals was 3.3.
After 48 hours: severe erythema (graded 4) with green/brown coloured necrosis over the whole application site was seen in 5/6 cases. In two of these 5 cases, small areas of blanching were seen and in one case, the reaction area was extended to 3 to 4 cm beyond the application site. The remaining animal showed well-defined erythema. The average score over all six animals was 3.7.
After 72 hours: severe erythema (graded 4) with green/brown coloured necrosis over the whole application site still was present in 5/6 cases. In the remaining animal, erythema turned to very slight (graded 2). The average score over all six animals was 3.5.
After 7 days: erythema still was severe (graded 4) in 5/6 animals and was accompanied by light brown-coloured eschar over the application site; in three of these 5 animals, pale yellow discoloration of the skin and/or fur also was seen. The remaining animal showed desquamation and pale yellow discoloration of the skin and/or fur. The average score over all six animals was 3.3.
After 14 days: at this time point, two animals showed a small area of slightly sunken brown-coloured eschar within the application site; no fur growth was observed within the remaining region of the application site. In one animal, a similar but more larger eschar area as described above was seen, with blood stained dermal tissue around the edge of the eschar. Furthermore, the skin showed a pale yellow discoloration and no fur growth was seen. Two further animals showed white scar tissue with no fur growth; in one case, this was accompanied by a pale yellow discoloration of the skin. One animal still showed desquamation and pale yellow discoloration of the skin and/or fur.
One hour after removal of the dressing: all animals displayed slight to moderate edema on the application sites (graded 2 to 3). The average score over all six animals was 2.8.
After 24 hours: four animals showed severe edema, which raised more than 1 mm and extended ventrally beyond the application site (graded 4). The remaining two animals showed slight to moderate edema (graded 2to 3). The average score over all six animals was 3.7.
After 48 hours: four animals showed moderate edema (graded 3) and one still showed severe edema, which raised more than 1 mm and extended ventrally beyond the application site (graded 4). The sixth animal was free from edema. The average score over all six animals was 2.7.
After 72 hours: three animals showed slight edema (graded 2) whereas two showed moderate edema (graded 3); the sixth animal still remained free of edema. The average score over all six animals was 2.0.
After 7 days: edema became slight in four cases (graded 2) and very slight in one case (graded 1); the sixth animal still was free of edema. The average score over all six animals was 1.5.
After 14 days: no more edema was seen.
Other effects:
A single, 4 hr semi-occluded dermal application of the test material to the skin of six rabbits produced severe dermal reactions, including eschar formation, necrosis and severe edema. Other adverse dermal reactions noted were slight haemorrhage of the dermal capillaries, blanching or brown discoloration of the skin, desquamation and scar tissue.The absence of fur growth was also occasionally noted on day fourteen.

Skin reaction

Reading (h)

Rabbit number

   

70

74

92

106  107  115 

Erythema/Eschar formation

1

3

3

1

24

4

4

2

   48  4
   7 days  4
   14 days  Su Su  0 DS  TS  SuS 
 Edema formation  1  3
   24  4
   48  3
   7 days  2
   14 days  0

Su = small area of slightly sunken brown-coloured eschar (approx. 5 mm x 5 mm). No fur growth over remainder of treatment site - corrosive effects noted.

S = pale yellow-coloured staining of skin/fur

D = desquamation

T = white scar tissue with no fur growth - evidence of corrosion

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The report in a compilation of data from studies conducted between 1958 and 1972. The data obtained from these studies are believed to be reliable despite of the fact, that the studies do not follow today's guideline requirements and that the test substance was not defined in terms of purity.
Principles of method if other than guideline:
The original study was conducted in 1972 according to the Draize Test; the assessment of the findings followed the FDA method(Federal Register, 37, No.83, 191.12, 1972). GLP was not compulsory at the time the studies were performed.
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Sex: female
Age/weight at study initiation: No data.
Vehicle:
other: PEG400
Controls:
yes, concurrent vehicle
Amount / concentration applied:
study TXM72065: Concentration: 0.5%, 2%, 5%; Amount applied: one drop
Duration of treatment / exposure:
study TXM72065: Treatment once/exposure 24 h
Observation period (in vivo):
study TXM72065: 21 days
Number of animals or in vitro replicates:
study TXM72065: 3 animals per group.
Details on study design:
TXM72065:
New Zealand White female rabbits were used for testing; each test group comprised 3 animals. Bronopol was tested as 0.5%, 2% and 5% solution in PEG 400; PEG 400 was tested in the control group. Each animal received a drop of test solution into the conjunctival sac of one eye; the second eye remained untreated and served as control. After 24 hours, the eyes were rinsed. The first reading of the eyes was performed after 1 hour; further reading time points were: 24 h, 48 h, 72 h, 7 d, 14 d and 21 d following treatment. Examination of the cornea at the time points 24 h, 72 h and 7 d was facilitated by instillation of one drop of fluorescein eye drop into the eye; the excess was flushed away with warm water. The assessment of the findings was based on the F.D.A method (Federal Register, 37, No. 83, 191.12, 1972).
Irritant / corrosive response data:
Study TXM72065, which was conducted 1972.This study was conducted according to Draize (i.e Draize test) and the assessment of the findings followed the F.D.A. method (Federal Register, 37, No. 83, 191.12, 1972). Following findings were reported:
Control (PEG 400) animals: At time point 1 hour, two control animals showed slight redness of the conjunctiva whereas slight to increased discharge was seen in all 3 control-animals. These effects disappeared within 24 hours. No chemosis was seen.
0.5% bronopol-treated animals: At time point 1 hour, all 3 animals showed slight redness of the conjunctiva; one of them also showed slight discharge; all these effect disappeared within 24 hours. No chemosis was seen.
2% bronopol-treated animals: At time point 1 hour, all 3 animals showed slight redness of the conjunctiva; this effect still was seen after 24 hours and in two cases, redness of the conjunctiva still was evident at time point 7 days following treatment. At time point 1 hour, all animals showed moderate to increased eye discharge; however, this effect disappeared within 24 hours. One case of chemosis was reported at time point 1 hour only.
5% bronopol-treated animals: Moderate redness of the conjunctiva was reported for all 3 animals at time point 1 hour; the redness lasted in all 3 animals up to time point 48 h. At time point 72 h, slight to moderate redness still was seen in 2 animals and after 7 days, in one of them. At time point 1 hour, all animals showed moderate to increased eye discharge; this effect lasted in all animals until time point 24 hours, but disappeared thereafter. Moderate to increased chemosis was seen in all 3 animals at time points 1 h and 24 h. At time point 48 h, chemosis remained in one animal; thereafter, no more chemosis was seen. All effects were reversible at least within 7 days. After 14 and 21 days, no more effects were seen. None of the concentrations elicited reactions in the cornea or iris.

Table 0: single animal data (extract from original table) after exposition to 5% bronopol

305 312 309 Mean (24,48,72 h)  
Score Conjunctiva redness 2 2 2 24 h
1 2 2 48 h 
1 2 0 72 h
Mean 1.3 2.0 1.3 1.6  
 
Score Conjunctiva swelling 2 3 2  24 h
0 1 0  48 h
0 1 0  72 h
Mean 0.7 1.7 0.7 1.0  
 
Score Conjunctiva discharge 1 1 2  24 h
0 0 0  48 h
0 0 0  72 h
Mean 0.3 0.3 0.7 0.4  

Table 1: Based on the F.D.A. scoring scale, the results for the different groups can be summarized as follows:

Treatment

Rabbit

Grade of irritation according to FDA scoring method

 

Control (vehicle: PEG 400)

1

negative

 

 2

negative

 3

negative

 0.5% Bronopol  1  negative
   2  negative
   3  negative
 2% Bronopol negative 
   2 negative 
   3 negative 
 5% Bronopol marginal 
   2 strongly irritated 
   3 irritant 
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation

Bronopol was tested for acute dermal irritation in the rabbit under semi-occlusive conditions (Safepharm Laboratories Limited,1987). The test group consisted of 6 New Zealand White rabbits. About 24 hours prior testing, the fur of the dorsal and flank area of each rabbit was clipped.The test material was prepared by moistening 0.5 g of test substance with 0.5 mL of distilled water and was applied on a selected shorn skin area, on the back of each rabbit. The application site was covered by means of a patch, which again was maintained in place using a strip of adhesive tape. Furthermore, the trunk of each rabbit was wrapped in a corset to prevent the animals from interfering with the patches.Exposure period was 4 hours; after removal of the dressing and the patch, residual test material on the application site of each rabbit was gently removed using cotton wool soaked in distilled water.The scoring of the dermal findings was performed according to Draize (1959); the reading time points were 60 min and 24 h, 48 h and 72 hours after removal of the patches. Additional readings were done on 7 and 14 days.

Erythema - one hour after removal of the dressing all animals displayed very slight to moderate - severe erythema on the application sites, slight haemorrhage of the dermal capillaries, brown discoloration and small areas of blanching were seen, which resulted in a scoring of 15. After 24 hours, erythema became severe in 4/6 cases and was accompanied by green/brown coloured necrosis over the whole application site.Small areas of blanching were seen in 5/6 animals, and slight hemorrhage of the dermal capillaries was seen in two of them.In two rabbits, the reaction area was extended to 3 to 4 cm beyond the application site. After 48 hours, severe erythema with green/brown coloured necrosis over the whole application site was seen in 5/6 cases.In two of these 5 cases, small areas of blanching were seen and in one case, the reaction area was extended to 3 to 4 cm beyond the application site.The remaining animal showed well-defined erythema. After 72 hours, severe erythema with green/brown coloured necrosis over the whole application site still was present in 5/6 cases. In the remaining animal, erythema turned to very slight. After 7 days, erythema still was severe in 5/6 animals and was accompanied by light brown-coloured eschar over the application site; in three of these 5 animals, pale yellow discoloration of the skin and/or fur also was seen. The remaining animal showed desquamation and pale yellow discoloration of the skin and/or fur. After 14 days, two animals showed a small area of slightly sunken brown-coloured eschar within the application site; no fur growth was observed within the remaining region of the application site.In one animal, a similar but larger eschar area as described above was seen, with blood stained dermal tissue around the edge of the eschar. Furthermore, the skin showed a pale yellow discoloration and no fur growth was seen. Two further animals showed white scar tissue with no fur growth; in one case, this was accompanied by a pale yellow discoloration of the skin. One animal still showed desquamation and pale yellow discoloration of the skin and/or fur.

Edema - one hour after removal of the dressing all animals displayed slight to moderate edema on the application sites. After 24 hours, four animals showed severe edema, which raised more than 1 mm and extended ventrally beyond the application site. The remaining two animals showed slight to moderate edema. After 48 hours, four animals showed moderate edema and one still showed severe edema, which raised more than 1 mm and extended ventrally beyond the application site. The sixth animal was free from edema. After 72 h, three animals showed slight edema whereas two showed moderate edema; the sixth animal still remained free of edema. After 7 d, edema became slight in four cases and very slight in one case; the sixth animal still was free of edema. After 14 days no more edema was seen. Conclusively, the test substance was highly irritating to the skin of rabbit.

This study is classified as acceptable (key study), because it was conducted according to the OECD guideline 404 (1981) in accordance with GLP (however, no data on test substance purity were given).

Supporting study:

In an in vitro study (BASF SE, 2008) the potential of Protectol BN(99% Bronopol) to cause dermal corrosion was assessed by a single topical application of 25μL bulk volume (about 41 mg) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm).Two EpiDerm tissue samples were incubated with the test substance for 3 minutes and 1 hour, each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as suitable endpoint. The formazan production of the test-substance treated epidermal tissues was compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability.A second experiment was performed, in which the test substance was treated under acidic conditions by moistening with 0.1 M Tris-HCl-buffer of pH 5 in order to enhance its stability in aqueous environment.Following results were obtained: The test substance was not able to directly reduce MTT. Viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 8 % (first experiment) and 26 % (second experiment). Viability of the test-substance treated tissues determined after an exposure period of 1 hour was 15% (first experiment) and 12 % (second experiment). The test substance shows a corrosive potential in the EpiDerm skin corrosivity test under both test conditions chosen.Nevertheless, the test method does not yet allow for the differentiation of severity of the effect.The results however indicate severe corrosivity. For final assignment of a risk phrase, further evidence or results from an in vivo study is needed.

This study is classified as acceptable (supporting study) because it is a GLP-conform in vitro study according to OECD GL 431.

Reference: Draize JH (1959). The appraisal of the safety of chemicals in foods, drugs and cosmetics.Association of Food and Drug Officials of the United States, Austin, Texas

Eye irritation

In an acute eye irritation/corrosion study (Knoll Pharmaceuticals, 1996) the irritating potential of Bronopol to the eye of rabbit was deemed. New Zealand White female rabbits were used for testing; each test group comprised 3 animals. Bronopol was tested as 0.5 %, 2 % and 5 % solution in PEG 400; PEG 400 was tested in the control group. Each animal received a drop of test solution into the conjunctival sac of one eye; the second eye remained untreated and served as control. After 24 hours, the eyes were rinsed. The first reading of the eyes was performed after 1 h; further reading time points were: 24, 48 and 72 hours, 7, 14 and 21 days following treatment; assessment of the findings was based on a F.D.A method.

Control animals: at time point 1 h, two control animals showed slight redness of the conjunctiva whereas slight to increased discharge was seen in all 3 control-animals. These effects disappeared within 24 hours. No chemosis was seen.

0.5% bronopol-treated animals: at time point 1 hour, all 3 animals showed slight redness of the conjunctiva; one of them also showed slight discharge; all these effect disappeared within 24 hours. No chemosis was seen.

2 % bronopol-treated animals: at time point 1 hour, all 3 animals showed slight redness of the conjunctiva; this effect still was seen after 24 hours and in two cases, redness of the conjunctiva still was evident at time point 7 d following treatment. At time point 1 hour, all animals showed moderate to increased eye discharge; however, this effect disappeared within 24 hours. One case of chemosis was reported at time point 1 h only.

5% bronopol-treated animals: moderate redness of the conjunctiva was reported for all 3 animals at time point 1 hour; the redness lasted in all 3 animals up to time point 48 h. At time point 72 h, slight to moderate redness still was seen in 2 animals and after 7 days, in one of them. At time point 1 hour, all animals showed moderate to increased eye discharge; this effect lasted in all animals until time point 24 h, but disappeared thereafter. Moderate to increased chemosis was seen in all 3 animals at time points 1 hour and 24 hours. At time point 48 hours, chemosis remained in one animal; thereafter, no more chemosis was seen. The grade of irritation were deemed as ‘marginal’ (rabbit 1), ‘strongly irritant’ (rabbit 2) and ‘irritant’ (rabbit 3) according to the F.D.A. scoring scale.

Considering the findings, Bronopol tested as 2 and 5 % solution in PEG 400 resulted in severe effects in rabbit eyes.

All observed effects were reversible within 14 days; after 14 and 21 days, no more effects were seen. None of the concentrations of the test substance elicited reactions in the cornea or iris.

Conclusively, Bronopol tested as solution in PEG 400 was slightly irritating.

This non-Guideline study was conducted in 1972 according to the Draize test; the assessment of the findings followed the F.D.A. method (Federal Register, 37, No. 83, 191.12, 1972). The data obtained from this study are believed to be reliable despite of the fact, that the study do not follow today's guideline requirements and that the test substance was not defined in terms of purity. Nevertheless, the study is classified as acceptable (key study).

 

Respiratory irritation

There are no direct experimental results available for this endpoint. However, in an acute inhalation study with the test substance (HazletonLaboratoriesEuropeLtd, 1986) it was clearly demonstrated that local irritation effects occur after inhalative exposure to Bronopol, e.g. nasal discharge or irritation of the eyes.


Justification for selection of skin irritation / corrosion endpoint:
The key study was selected.

Justification for selection of eye irritation endpoint:
The key study was selected.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Effects on respiratory irritation: irritating

Justification for classification or non-classification

Based on the results for in vivo skin irritation, Bronopol is classified as Xi, R38 (irritating to skin) according to the criteria of Directive 67/548/EEC and as Skin Irrit.2, H315 (causes skin irritation) according to Regulation (EC) No 1272/2008 (self-classification).Furthermore, a corrosive potential of the test substance was revealed in the EpiDerm skin corrosivity test.

Based on the results of the in vivo eye irritation study, the test substance is not classified as eye irritant according to Directive 67/548/EEC or to EU Regulation (EC) No 1272/2008.

Due to findings of local irritations in an acute inhalation toxicty test, the test substance was tagged with Xi, R37 according to Directive 67/548/EEC and STOT SE3; H335 according to Regulation (EC) No 1272/2008 (self-classification).

 

Bronopol is officially classified as R41 (risk of serious damage to eyes) and Eye Dam. 1, H318 (causes serious eye damage) according to Directive 67/548/EEC and Regulation (EC) No 1272/2008, respectively.

Furthermore, Bronopol is officially classified as Xi, R37/38 (irritating to respiratory system and skin) and Skin Irrit. 2, H315 (causes skin irritation) and STOT SE 3, H335 (may cause respiratory irritation) according to Directive 67/548/EEC and Regulation (EC) No 1272/2008, respectively.