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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:
8 JUL 1996 - 9 OCT 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed in compliance with the Good Laboratory Practice (GLP) regulations. The method followed that described in the OECD Guidelines for Testing of Chemicals, Updated Guideline No 406 Skin Sensitisation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Guinea pig maximisation test was performed before the LLNA was set as preferred test method.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
425-560-7
EC Name:
-
Cas Number:
174063-87-7
Molecular formula:
C33H32O10
IUPAC Name:
2-methylbenzene-1,4-diyl bis{4-[3-(acryloyloxy)propoxy]benzoate}
Test material form:
solid: crystalline

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: HsdPoc:DH
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Guinea pig, CRL:(HA)BR, females
- Source: Charles River Kißlegg
- Age at study initiation: about 6 weeks
- Weight at study initiation: 381 g (range from 344 to 416 g)
- Housing: Two guinea-pigs were housed in a Makrolon cage type IV (floor area: 55 x 33 cm = 1815 cm2, height: 20 cm) placed on mobile racks. The animals were kept on conventional softwood granulate as bedding. The cages had been machine-cleaned before the start of the study. The bedding was changed three times a week.
- Diet (e.g. ad libitum): ad libitum (This diet is checked periodically by an independent and German Government approved laboratory, according to the specifications of the manufacturer. Analysis includes both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides and antibiotics.)
- Water (e.g. ad libitum): ad libitum (community tap water from Makrolon drinking bottles, regularly analyzed microbiologically, physicochemically, and chemically)
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 26
- Humidity (%): 37 - 71
- Photoperiod (hrs dark / hrs light): 12/12

Remark: The room temperature and relative atmospheric humidity in the animal room transiently exceeded the target range of 20 to 24°C and 45 to 75%. These small and short deviations did not influence the result of the study.







Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
Intradermal injection of test material preparation (1.0 g/L with and without Freund's complete adjuvant) on day 1, and topical application of test material preparation (50.0 g/L) on day 8.
Day(s)/duration:
Day 1 (intradermal), Day 8 (topical)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
Topical application the test material preparation (10.0 g/L) was performed two weeks after topical induction.
Day(s)/duration:
Day 22
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
Due to the questionable results after the first challenge (challenge I), a second challenge (challenge II) with a lower concentration of the test material (1.0 g/L) was considered to be necessary, two weeks later.
Day(s)/duration:
Day 36
Adequacy of challenge:
other: non-irritant
No. of animals per dose:
Total: 33 females
Pre-test: 3 females
Control group: 10 females
Test group 20 females
Details on study design:
RANGE FINDING TESTS:
To determine the concentrations suitable for the main study, a pretest with single intradermal or topical administrations of the vehicle and of the test item preparations was performed. Intradermal (i.d.) injections were given to one animal. Topical (top.) applications were given to another two animals, one with and one without FCA injection.
The following concentrations were used:
1. Liquid paraffin (vehicle)
intradermal: undiluted
2. test item
intradermal: 10, 5, and 1 g/L paraffin;
topical: 250, 100, 50, and 10 g/L paraffin
topical with FCA: 10, 5, and 1 g/L paraffin

MAIN STUDY

A. INDUCTION EXPOSURE
- No. of exposures: 2 (injection and topical application)
- Exposure period: 48 h (topical application)
- Test groups:
1. intradermal induction:
cranial: 0.05 ml Freund's complete adjuvant + 0.05 ml phys. sodium chloride solution
medial: 0.10 ml test item (1g/L liquid paraffin)
caudal: 0.05 ml Freund's complete adjuvant with test item + 0.05 ml phys. sodium chloride solution (1.0 g/L completed preparation)
2. topical induction: test item (50.0 g/L liquid paraffin)

- Control group:
1. intradermal induction:
cranial: 0.05 ml Freund's complete adjuvant + 0.05 ml phys. sodium chloride solution
medial: 0.10 ml liquid paraffin
caudal: 0.05 ml Freund's complete adjuvant + 0.05 ml phys. sodium chloride solution
2. topical induction: liquid paraffin (undiluted)

- Site:
1. injection induction: shoulder region (cranial, medial, caudal)
2. topical induction: shoulder region

- Frequency of applications: weekly
- Duration: 48 h (topical induction)
- Concentrations:
1. intradermal induction:
0.10 mL test item (1g/L liquid paraffin)
2. topical induction:
1 mL test item (50.0 g/L liquid paraffin)

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 14 (challenge 1), 28 (challenge 2)
- Exposure period: 24 h
- Test groups, Control group:
Two or four weeks after the topical induction the challenge was performed by fixing filter papers of about 4 cm2 fully loaded with 0.5 ml of the test item preparations or soaked with 0.5 ml liquid paraffin to the shaven flank of the animals. The patches were fixed for 24 hours with occlusive plastic tape.
- Site: flank
- Concentrations:
Challenge 1: 0.5 mL of test item (10 g/L)
Challenge 2: 0.5 mL of test item (1 g/L)
- Evaluation (hr after challenge): 48 and 72 h after start of challenge

OTHER:
MAIN STUDY-METHOD

+ ++ I II
/__________________/__________________/__________________/__________________/__________________/
Study day: 1 8 22 36

induction: + = intradermal injection
++ = topical application
challange: I + II = topical challange


-- Observations
The body weight of the guinea pigs was determined prior to the start of the study then on day 8, 15,22, 29, 36 and at the end of the study.

After the challenge, skin changes at the application sites were evaluated according to the following MAGNUSSON and KLIGMAN scheme.

Following the grading according to the EEC Directive 91/325, a result in an adjuvant method is considered positive if 30% or more of the test animals.
Challenge controls:
yes (vehicle only)
Positive control substance(s):
yes
Remarks:
1-chloro-dinitrobenzene, historical data

Results and discussion

Positive control results:
The sensitivity of the test system is demonstrated periodically with 1-Chloro-2,4-dinitrobenzene (DNCB), a known sensitiser.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
1.0 g/L (induction), 10 g/L (challenge)
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
no signs of toxicity
Remarks on result:
other: challenge I
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
1.0 g/L (induction), 10 g/L (challenge)
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
positive reactions in the treated areas after challange, scales were seen in the treated skin areas of 16 animals
Remarks on result:
other: challenge I
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
1.0 g/L (induction), 1.0 g/L (challenge)
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
positive reactions in the treated areas after challange
Remarks on result:
other: challenge II
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
1.0 g/L (induction), 1.0 g/L (challenge)
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
positive reactions in the treated areas after challenge
Remarks on result:
other: challenge II
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
no induction, 10 g/L (challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
other: challenge I
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
no induction, 10 g/L (challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
6 animals showed scales in the skin area treated
Remarks on result:
other: challenge I
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
no induction, 1.0 g/L (challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
1 animal showed scales in both treated areas (test item and paraffin treated)
Remarks on result:
other: challenge II
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
no induction, 1.0 g/L (challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
other: challenge II
Group:
positive control
Remarks on result:
not measured/tested
Remarks:
The sensitivity of the test system is demonstrated periodically with 1-Chloro-2,4-dinitrobenzene (DNCB), a known sensitizer.

Any other information on results incl. tables

Pre-test data

Vehicle: Liquid paraffin (undiluted)

intradermal induction: 1.0 g/l slightly irritant

topical induction: 50.0 g/l slightly irritant

topical challenge: 10.0 g/l not irritant

Findings in the induction phase

After intradermal injection, the common signs of irritation after injection of Freund's complete adjuvant were seen. The injection sites were swollen and red, later on necroses and scabs developed. Similiar reactions were seen after injection with liquid paraffin.

After removal of the patches, swollen and red application sites and scabs were observed at the application sites.

Findings after challenge I

The first challenge was performed to the right flank of the animals.

Group 1: negative control group

After a single treatment with liquid paraffin or with the test material preparation on separate patches each, no erythema or edema were observed at the two readings respectively. But 72 hours after exposure to the preparation, six animals showed scales in the treated areas. One animal, treated with liquid paraffin, showed scales 72 hours after exposure.

Group 2: test material group

Challenge was performed with liquid paraffin to exclude sensitisation to the vehicle; and no positive reactions in the treated areas were observed at any reading. Topical challenge, as a preparation of 10.0 g/l was performed . Positive reactions were observed in 7 of 20 animals (35 %) at both readings. Scales were seen in the treated skin areas of 16 animals at the second reading.

Findings after challenge II

The second challenge was performed to the left flank of the animals.

Group 1: negative control group

Single treatment on the left flank with liquid paraffin or with a preparation on separate patches each, did not lead to any erythema or edema. Only one animal showed scales in both treated areas at the first reading.

Group 2: test material group

A challenge was performed with liquid paraffin to exclude sensitisation to the vehicle; and no erythema or edema were observed in the treated areas. Topical challenge, as a test item preparation of 1.0 g/l, was performed. After challenge, positive reactions in the treated areas were observed in 19 of 20 animals (95 %) at both readings. Scales were seen in 16 animals at the second reading.

Overall the controls gave no unwanted effects and the results of the sensitsation rate increased from 7/ 20 animals (35%) at the first reading to 19/ 20 animals (95%) at the second challenge.

Clinical findings and mortality

The clinical behaviour of the guinea pigs was normal during the experimental part. All animals survived the experimental part.

Body weight

The body weight development corresponded to that of the animals of the vehicle group.

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
According to MAGNUSSON and KLIGMAN (1969) the test material has to be classified as a skin sensitiser. This classification corresponds to a sensitisation rate of 81 to 100 %. According to the Regulation (EC) No 1272/2008, the test material has to be classified as a skin sensitiser category 1A.
Executive summary:

The registered substance was tested for skin sensitisation in a guinea pig maximisation test according to OECD Guideline 406 following GLP.

Purpose
The purpose of this GPMT assay was to identify the contact allergenic potential of the test item. This study should provide a rational basis for risk assessment to the sensitising potential of the test item in human.

Study Design
The test item was investigated for skin sensitising properties in the guinea pig maximization test according to MAGNUSSON and KLIGMAN (1969).

10 female guinea-pigs in the negative control group (group 1) and 20 females in the test material group (group 2) were investigated.

Induction included intradermal injection of test material preparation (1.0 g/l with and without Freund's complete adjuvant) on day 1, and topical application of test material preparation (50.0 g/l) on day 8.

Challenge by topical application the test material preparation (10.0 g/l) was performed two weeks after topical induction. Due to the questionable results after the first challenge (challenge I), a second challenge (challenge II) with a lower concentration of the test material (1.0 g/l) was considered to be necessary, two weeks later.

Results

Challenge I  (10.0 g/L)

Group challanged with positive / total
(after start of challange)		 positive / animals
48 h 72 h overall
1 paraffin 0/10 0/10 0/10
Control test material 0/10 0/10 0/10
2 test material 6/20 1/20 7/20
paraffin 0/20 0/20 0/20



Challenge II  (1.0 g/L)

Group challanged with positive / total
(after start of challange)		 positive / animals
48 h 72 h overall
1 paraffin 0/10 0/10 0/10
Control test material 0/10 0/10 0/10
2 test material 18/20 8/20 19/20
paraffin 0/20 0/20 0/20



Challenge I (on the right flank)
Group 1: negative control group
After a single treatment with liquid paraffin or with a preparation on separate patches each, no erythema or edema were observed. But 72 hours after exposure, six animals showed scales in the skin area treated with the preparation. One animal, treated with liquid paraffin, showed scales 72 hours after exposure.

Group 2: test material group
Topical challenge was performed with liquid paraffin to exclude sensitisation to the vehicle and no positive reactions in the treated areas were observed. Topical challenge was performed with a preparation of 10.0 g/l . Positive reactions (erythemas) were observed in 7 of 20 animals (35 %). Scales were seen in the treated areas of 16 animals at the second reading.

Challenge II (on the left flank)
Group 1: negative control group
Single treatment with liquid paraffin or with a preparation on separate patches each, did not lead to positive reactions of the skin. Only one animal showed scales in both treated areas and only at the first reading.

Group 2: test material group
A challenge was performed with liquid paraffin to exclude sensitisation to the vehicle; and no positive reactions in the treated areas were observed at the following readings. Topical challenge was performed with a test item preparation of 1.0 g/l . After challenge, positive reactions in the treated areas were observed in 19 of 20 animals (95 %). Scales were seen in 16 animals at the readings.

Overall, the controls had no unexpected effects and the results of the sensitsation rate increased from 35% at the first challenge to 95% at the second challenge.

Conclusion
According to MAGNUSSON and KLIGMAN (1969) the test material has to be classified as an extreme sensitiser. This classification corresponds to a sensitisation rate of 81 to 100 %. According to the Regulation (EC) No 1272/2008 the test material has to be classified as a sensitiser.