Registration Dossier

Administrative data

Description of key information

Bronopol caused skin irritation in a study on rabbits.

Bronopol caused irreversible damage to the eye in a study on rabbits.

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
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 or test system 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

Interpretation of results:
Category 2 (irritant) based on GHS criteria
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
Study period:
28 Aug 2000 - 8 Nov 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
Himalayan
Details on test animals or tissues and environmental conditions:
Stock/Breeder: LPT Laboratory of Pharmacology and Toxicology KG, branch Lohndorf, D-24601 Lohndorf/Post Wankendorf
Sex: male
Age: 8.5-9mos
Weight: 2.7 kg
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
100 mg
Duration of treatment / exposure:
single exposure
Observation period (in vivo):
Examined at 60 min, 1, 24, 48, 72 hours, daily from day 4-21
Number of animals or in vitro replicates:
1
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: 1 h
Score:
4
Max. score:
4
Reversibility:
other: further assessment not possible
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: 1 h
Score:
2
Max. score:
2
Reversibility:
other: further assessment not possible
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.3
Max. score:
2
Reversibility:
not reversible
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3.6
Max. score:
4
Reversibility:
not reversible
Irritant / corrosive response data:
Cornea: Grade 4 opacity was observed at 1 hour after application. Further assessment was not possible: whitish deposits (probably pus) were seen from 72 hours and eyelids sticking together from day 5 after application onwards. The cornea was completely destroyed by day 18 after treatment.
Iris: Grade 2 irritation of the iris was observed at 1 hour after application. Further assessment of the iris was not possible.
Conjunctiva:
Redness: Grade 1-2 conjunctival redness was seen during the complete observation period except for days 5-17, where no assessment was possible.
Chemosis: Grade 3-4 conjunctival chemosis was seen between 1 hour and 18 days after application. Afterwards grade 2 chemosis was observed.
Other effects:
Systemic intolerance reactions were not observed.

 

Cornea

Iris

Conjunctiva

redness

chemosis

score (average of animals investigated)

0 to 4

0 to 2

0 to 3

0 to4

60 min

4

2

2

3

24 h

n.p.

n.p.

1

3

48 h

n.p.

n.p.

1

3

72 h

n.p.

n.p.

2

4

Average 24h, 48h, 72h

n.p.

n.p.

1.3

3.3

Day 21

Cornea destroyed

-

1

2

Area effected

Not reported

Maximum average score (including area affected, max 110)

-

-

-

-

Reversibility*

n

n

n

n

average time for reversion

-

-

-

-

 

*            c :          completely reversible
              n c :      not completely reversible
              n :         not reversible

-

-

-

-

n.p. = assessment not possible

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Reactions in the cornea, iris and the conjunctiva revealed severe irritation of the eye. The cornea was completely destroyed by day 18 after treatment. Eye reactions were not reversible within 21 days after application.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

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 studies:

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. This study was not considered for classification since the absence of corrosive effects of bronopol was confirmed in an in vivo study.

 

Bronopol was tested for dermal irritation / corrosion in another study in rabbit under semi-occlusive conditions (Lanxess, 2000). The test group consisted of 3 Himalayan rabbits. 500 mg Bronopol was applied to a shaved test site of approximately 6 cm2. The exposure was conducted semiocclusive using a patch coverage. Exposure period was 24 hours. 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 patch. There were no skin reactions in any rabbit at any of the observation points. The average score for erythema and edema was 0. Under the conditions of the test, the test material was not irritating to the skin of rabbit.

This study is classified as acceptable (supporting study), because it was conducted according to the OECD guideline 404 and in accordance with GLP. However, applying a conservative approach, this study was not used for classification, since bronopol was detrrmined to be irritating to the skin in study by Safepharm Laboratories Limited (1987).

 

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

Bronopol was tested for eye irritation in a GLP compliant study one Himalayan rabbit (Lanxess, 2000) according to OECD test guideline 405. 100 mg were instilled in the eye. Eye irritation was examined at 1, 24, 48 and 72 hours, and daily from day 4-21. Grade 4 opacity was observed at 1 hour after application. Further assessment was not possible: whitish deposits (probably pus) were seen from 72 hours and eyelids sticking together from day 5 after application onwards. The cornea was completely destroyed by day 18 after treatment. Iris: Grade 2 irritation of the iris was observed at 1 hour after application. Further assessment of the iris was not possible. Conjunctiva Redness: Grade 1-2 conjunctival redness was seen during the complete observation period except for days 5-17, where no assessment was possible. Chemosis: Grade 3-4 conjunctival chemosis was seen between 1 hour and 18 days after application. Afterwards grade 2 chemosis was observed. Systemic intolerance reactions were not observed. Reactions in the cornea, iris and the conjunctiva revealed severe irreversible eye damage.

This study is classified as acceptable (key study), because it was conducted according to the OECD guideline 405 and in accordance with GLP.

Supporting study:

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 an 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. However, since only a 5% suspension of bronopol was tested this study cannot be used for classification. Nevertheless, the study is classified as acceptable as supporting 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. However, symptoms of local irritation only occured at concentrations causing lethality.

 

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: serious eye damage

Effects on respiratory irritation: not irritating

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is considered to be classified for skin irritation Cat. 2A and serious eye damage Cat. 1 under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

Furthermore, Bronopol is included in Annex VI of Regulation (EC) No. 1272/2008 with the following legal classification:

  • Skin irritation Cat. 2 (H315: causes skin irritation)
  • Eye Damage Cat. 1 (H318: causes serious eye damage)
  • STOT SE Cat. 3 (H335: May cause respiratory irritation).