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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 3rd and October 4th, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study report does not mention the CAS number or contain the Certificate of Analysis of the test substance. However, this GLP study was conducted per the then-existing OECD 406 (year 1981) and EEC B6 (year 1984) test guidelines and has used an approved scientific methodology (Magnusson and Kigman, 1969) which satisfies basic scientific requirements of the test.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
test conducted before adoption of LLNA method

Test material

Constituent 1
Reference substance name:
Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP)
IUPAC Name:
Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP)
Constituent 2
Chemical structure
Reference substance name:
Bis(4-methylbenzoyl)peroxide
EC Number:
407-950-9
EC Name:
Bis(4-methylbenzoyl)peroxide
Cas Number:
895-85-2
Molecular formula:
C16H14O4
IUPAC Name:
4-methylbenzoyl 4-methylbenzene-1-carboperoxoate
Test material form:
other: Solid, white
Details on test material:
- Name of test material (as cited in study report): Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP)
- Physical state: Solid, white
- Analytical purity: Water damped powder 72% PMBP
- Batch No.: IR 240001
- Stability of test article: Stable
-Expiration date: November, 1991
- Stability of test article dilution: Stable for at least 48 hours in acetone; stability in vaselinum album is excluded from Statement of Compliance
- Storage condition of test material: In the original container; protected from light at room temperature (approx. 20 degrees C)

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted)
Sex:
female
Details on test animals and environmental conditions:
Rationale: Recognized by the international guidelines as the recommended test system, (e.g. OECD, EEC).
Source: BRL, Biological Research Laboratories Ltd. Wolferstrasse 4, CH-4414 Fullinsdorf
Number animals for main study/pretest: 30 females / 6 females
Age at acclimatization start: 4 - 7 weeks
Body weight at acclimatization start: Control and test group: 298 - 409 g; Pretest: 297 - 308 g
Identification: By unique cage number and corresponding ear tags.
Randomization: Randomly selected at time of delivery.
Acclimatization: One week under test conditions after veterinary examination.

Standard Laboratory Conditions
Air-conditioned with 10-15 air changes per hour and continuously monitored environment with a temperature of 22 ± 3 degrees centigrade, a relative humidity between 40-70 %, 12 hours artificial fluorescent light/12 hours dark, music during the light period.

Accommodation
Individually in Makrolon type-3 cages with standard softwood bedding ("Lignocelli, Schill AG, CH-4132 Muttenz).

Diet
Pelleted standard Kliba 342, Batches 63/91 and 64/91 guinea pig breeding/maintenance diet ("Kliba", Klingentalmuhle AG, CH-4303 Kaiseraugst), ad libitum. Results of analysis for contaminants were included in the Study Report.

water
Community tap water from Fullinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/L) via the drinking water. Results of bacteriological, chemical and contaminant analyses were included in the Study Report.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Acetone for induction (intradermal); vaselinum album for epidermal and challenge applications.
Concentration / amount:
Induction:
Intradermal injection: 0.1 ml per site (prepared as described under study design)
Epidermal application: 15% in vaselinum album
challenge: 5% in vaselinum album
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Acetone for induction (intradermal); vaselinum album for epidermal and challenge applications.
Concentration / amount:
Induction:
Intradermal injection: 0.1 ml per site (prepared as described under study design)
Epidermal application: 15% in vaselinum album
challenge: 5% in vaselinum album
No. of animals per dose:
As described under study design.
The animals were distributed as follows: 10 females for the control group and 20 females for the test group. Two females were used for the intracutaneous pretest and 4 females for the epicutaneous pretest.

FEMALE GROUPS ANIMAL NUMBERS

1 Control Group 331 - 340

2 Test Group 341 - 360

3 i.c. Pretest 361 362

4 e.c. pretest 363 – 366
Details on study design:
STUDY CONDUCT. TREATMENT PROCEDURE - PRETEST
The objective of this investigation was to identify irritant test article concentrations suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application. The procedure employed for these investigations was as follows:

Intradermal injections: Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1% of the test article in acetone. The resulting dermal reactions were assessed 24 hours later. For intracutaneous induction application a 1% test article dilution was selected.

Epidermal applications: Patches of filter paper (2 x 2 cm) were covered with a thin layer of the test article in vaselinum album at concentrations of 25, 15, 10 and 5% and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24 hours and the reaction sites were assessed for erythema and edema on a numerical basis according to Draize described above. Examination of the sites were performed 24 and 48 hours after removal of the dressings.

The position of the epidermal applications is shown below:

cranial
A C
B D
............
caudal

The allocation of the different test sites on the animals was alternated in order to minimize site to site variation in responsiveness.
MAIN STUDY
Induction

Intradermal injections: An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:

Test group:
1) Freund's complete adjuvant 50:50 with physiological saline.
2) The test article, diluted to 1% with acetone.
3) The test article diluted to 1% by emulsion in a 50:50 mixture of acetone and Freund's complete adjuvant:physiological saline (1:1).

Control Group:
1) Freund's complete adjuvant 50:50 with physiological saline.
2) Acetone.
3) 50:50 mixture of acetone and Freund's complete adjuvant:physiological saline (1:1).

The positions of the intradermal injections are shown below:

Cranial
Left Right
1 1
2 2
3 3
Caudal

1,2,and 3 are spots in which injections were made. The larger clipped area includes the area of injections shown above.

Epidermal applications: One week after the injections, the scapular area (approximately 6x8 cm) was again clipped and shaved free of hair. A 2x4 cm patch of filter paper was covered with a thin layer of the selected test article concentration (15% in vaselinum album) and placed over the injection sites of the test animals. The patch was covered by aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hrs. This application procedure ensured intensive contact of the test article.

The guinea pigs of the control group were treated as described above with the omission of test article (vaselinum album only).

Reaction sites were assessed for erythema and edema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

First Challenge
The test and control guinea pigs were challenged two weeks after the epidermal induction application. Hair was clipped and shaved from a 5 x 5 cm area on theleft and right flank of each guinea pig. Two patches (2 x 2 cm) of filter paper were covered with a thin layer of a a) non-irritant concentration of the test article (5% in vaselinum album and b) with vaselinum album only and applied to the a) left flank and b) right flank using the same method as for the epidermal application. The dressings were removed approximately 24 hours later. The sites were assessed for erythema and edema 24 and 48 hours after removal of the dressing, using the numerical scoring system according to Draize.

The control animals were treated in the same way as described above. Erythema and edema reactions are described in the tables under Appendix A.

OBSERVATIONS
Reading of challenge reactions: The challenge site was evaluated 24 and 48 hours after removal of the patch. The readings were made under artificial fluorescent light (daylight spectrum). Redness constitutes the minimum criterion of an allergic reaction. Strongly sensitized animals display a vivid redness, associated with indurated swelling. The reactions were scored on the basis of the Draize score described below:

READINGS AND SCORING: The following parameters were recorded.

Erythema (E) 0 to 4 numerical scores
Edema (Oe) 0 to 4 numerical scores
Diameter (D) mm

Erythema and edema were assessed using the following numerical grading system according to Draize:

Erythema and eschar formation:
Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to slight
eschar formation (injuries in depth) 4

Edema formation:
Score
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well-defined by definite raising) 2
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised more than 1 millimeter and
extending beyond the area of exposure) 4

Rating of allergenicity: Based upon the percentage of animals sensitized (24-hour reading), the test article was assigned to one of the following five grades of allergenic potency, ranging from weak to extreme.

Sensitization Rate Grade Classification
[%]
0 - 8 = 1 ....weak
9 - 28 =2 ....mild
29 - 64 =3 ....moderate
65 - 80 =4 ....strong
81 - 100 =5 ....extreme

Interpretation:
The results obtained in test animals at the challenge applications were compared with the results obtained in control animals. An allergic reaction was defined by visible reddening of the challenge site. The significance of difference in the number of animals with positive reactions between the treated group and the control group was assessed by the Fisher Test. If the dermal reactions of test animals at challenge were more marked and/or persistent than those of the control animals, the animals were considered to show evidence of contact hypersensitivity. If the dermal reactions of test animals at challenge were not clearly different from the reactions in control group animals, the results for the test animals were considered "inconclusive". If the dermal reactions to the challenge application were identical to or less marked and/or persistent than the dermal reactions in control animals, the test animals were considered to show no evidence of contact hypersensitivity.

In addition to the sensitizing reactions, the following observations and data were recorded during the test and observation period:

Viability/Mortality ...........Daily
Body Weights ...........At acclimatization start, at day one and at termination of test
Clinical Signs (local/systemic) ...........Daily
Skin reactions ...........At the time of reactions readings during induction and challenge period.

Records were maintained on all additional and standard observations.

PATHOLOGY
No necropsy was performed in the animals euthanized at termination of observation. All animals were euthanized at the end of the test period with an intraperitoneal injection of Pentobarbital (>800 mg/kg) and discorded.

STATISTICAL ANALYSIS
Mean values with standard deviations.
Fisher-Test (The Exact Fisher Test for comparison of the basic probability of two binomial distributions. L. Sachs, Statistische Auswertungsmethoden, Georg Thieme Verlag, Stuttgart 1971).
For calculation of p-values, the 24-hour reading of the animals from the control and test group was used.

DATA COMPILATION
The following data were recorded on data sheets and transcribed for compilation and analysis: skin reactions, viability/mortality, and clinical signs (local/systemic). Body weights were recorded on-line.
Challenge controls:
As described under Study Design (above)
Positive control substance(s):
yes
Remarks:
For the induction period a 20% (a.i.) dilution of FORMALDEHYDE SOLUTION (HCHO) in bi-distilled water and for the challenge procedure a 15% (a.i.) dilution of FORMALDEHYDE SOLUTION (HCHO) in bi-distilled water was used.

Results and discussion

Positive control results:
POSITIVE CONTROL: FORMALDEHYDE - SOLUTION (HCHO):

For the induction period a 20% (a.i.) dilution of FORMALDEHYDE SOLUTION (HCHO) in bi-distilled water and for the challenge procedure a 15% (a.i.) dilution of FORMALDEHYDE SOLUTION (HCHO) in bi-distilled water was used. The positive control article FORMALDEHYDE SOLUTION (HCHO) was delivered by Fluka AG, 9470 Buchs, Switzerland (Article No. 4003) and the purity was described to be at least 37% stabilised with about 10% methanol.

POSITIVE ERYTHEMA REACTIONS AFTER FIRST CHALLENGE PROCEDURE
-----------------------------------------------------------
after 24 hours positive / total: 8 / 10
% positive out of total: 80

According to the procedures used in this experiment (run from June 17 to July 16, 1991), clear positive results were observed in the (HCHO) treated animals after the epidermal challenge application. According to the results observed it is considered that the known allergen FORMALDEHYDE SOLUTION (HCHO) possess a strong skin sensitizing potential in the guinea pig strain used (Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted); BRL, Biological Research Laboratories Ltd., CH-4414 Fullinsdorf).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
other: control group
Dose level:
vaselinum album (right flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: control group. Dose level: vaselinum album (right flank). No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: control group
Dose level:
vaselinum album (right flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: control group. Dose level: vaselinum album (right flank). No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: control group
Dose level:
Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP (5%), left flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: control group. Dose level: Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP (5%), left flank). No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: control group
Dose level:
Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP (5%) (left flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: control group. Dose level: Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP (5%) (left flank). No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
vaselinum album (right flank)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: vaselinum album (right flank). No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vaselinum (right flank)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: vaselinum (right flank). No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP (5%) (left flank)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP (5%) (left flank). No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP (5%) (left flank)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP (5%) (left flank). No with. + reactions: 0.0. Total no. in groups: 20.0.

Any other information on results incl. tables

PRE TEST

RESULTS

 

During pretest, the following reactions were observed.

 

INTRADERMAL INJECTION

 

Vehicle: Acetone

 

 

Animal number

Sex

Concentration %

REACTION READINGS AFTER 24 HOURS

Erythema

Oedema

Diameter

[mm]

361

F

5

2

1

10x10

362

F

5

2

1

11x10

361

F

3

2

1

9x9

362

F

3

2

1

8x10

361

F

1

1

1

6x7

362

F

1

1

1

8x8

 

According to Magnusson - Kligman and to the findings observed, the concentration selected for the main study was 1%.

 

EPIDERMAL APPLICATION

 

Vehicle: Vaselinum album

 

 

 

 

REACTION READINGS AFTER REMOVAL OF BANDAGE

 

 

 

after 24 hours

after 48 hours

Animal number

Sex

Concentration %

E

Oe

E

Oe

363

F

25

15

10

5

1

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

364

F

25

15

10

5

0

1

1

0

0

0

0

0

0

1

1

0

0

0

0

0

365

F

25

15

10

5

0

0

1

0

0

0

0

0

0

0

1

0

0

0

0

0

366

F

25

15

10

5

1

1

0

1

0

0

0

0

1

0

0

1

0

0

0

0

 

 

E = Erythema

Oe = Oedema

 

According to Magnusson - Kligman, and to the findings observed, the concentration selected for the induction period was 15% and for the challenge procedure was 5%.

Main Study

SENSITIZING EFFECTS

CONTROL GROUP/ TEST GROUP: No positive reactions, scores where 0, were evident after the first challenge application, neither when treated with vaselinum album only nor when treated with a 5% test article dilution.

 

VIABILITY/MORTALITY: No death occurred.

 

CLINICAL SIGNS, LOCAL

CONTROL GROUP / TEST GROUP: Application area around the injection sites 1, 2 and 3 was found to show erythema and edema from day 2 to 6 of test, necrosis from day 7 to 11, encrustation from day 12 to 17 and exfoliation from day 18 to 25 (termination of test). On day 9 of test no observation could be performed because the animals were bandaged semi-occlusively.

 

CLINICAL SIGNS, SYSTEMIC

No systemic symptoms were observed in the animals.

 

BODY WEIGHTS

Three out of 20 test females and 2 out of 4 animals of e.c. pretest lost weight during the acclimatization period. The body weight gain of the other animals was not affected adversely during the study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
From the results described above no allergenic potency of the test article Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP) was concluded. The results were interpreted according to the rating of Magnusson and Kligman (1969). According to EEC (European Economic Community) classification criteria described in guidelines 83/467, September 16, 1983 and 67/548, May 1987, this test article is considered not to be a sensitizer.
Executive summary:

SUMMARY

To assess the allergenic potential of Di-(4 -Methylbenzoyl)-peroxid (INTEROXPMBP) in albino guinea pigs the Maximization Test of B. Magnusson and A.M. Kligman (1969) was used by administering the test substance, Di-(4 -Methylbenzoyl)-peroxid (INTEROX-PMBP) to the skin of guinea pigs.

This test should provide a rational basis for risk assessment of the sensitizing potential of the test substance.

Ten females were used as control group and 20 females were used as test group. The study was conducted between September 3rd and October 4th, 1991 at the BRL laboratories in CH-4414 Fullinsdorf. A reliability rating of K2 was given due to the non-inclusion of the CAS number and the Certificate of Analysis of the test substance with the study report. However, this GLP study was conducted per the then-existing OECD 406 (year 1981) and EEC B6 (year 1984) test guidelines and has used an approved scientific methodology which satisfies basic scientific requirements of the test.

RESULTS:

Clear positive results were observed in the positive control (HCHO) treated animals after the epidermal challenge application.

In the actual test, no toxic symptoms were evident in the guinea pigs of the control or test group. No death occurred.

The highest non-irritating concentration of the test substance (CAS 895 -85 -2) used for challenge application was 5%.

Due to the unequivocal findings observed after the first challenge, no second challenge was performed.

 

POSITIVE ERYTHEMA REACTIONS AFTER FIRST CHALLENGE PROCEDURE WERE AS FOLLOWS:

 

 

 

 

 

After 24 hours:

  positiveItotal

% positive of total

After 48 hours:

   positiveItotal

% positive of total

CONTROL GROUP

Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP) (left flank) (left flank)

0/ 10

0%

0/ 10

0%

 

vaselinum album (right flank)

0/ 10

0%

0/ 10

0%

TEST GROUP

Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP) (left flank)

0I 20

0%

0/ 20

0%

 

vaselinum album (right flank)

0/ 20

0%

0/ 20

0%

CONCLUSION: 

From the results described above no allergenic potency of the test article Di-(4-Methylbenzoyl)-peroxid (INTEROX-PMBP) was concluded. The results were interpreted according to the rating of Magnusson and Kligman (1969). According to EEC (European Economic Community) classification criteria described in guidelines 83/467, September 16, 1983 and 67/548, May 1987, this test article is considered not to be a sensitizer.