Dimethyl (p-methoxybenzylidene)malonate

Endpoint summary

Description of key information

No sensitisation was observed in a GLP-compliant guinea pig maximisation test according to OECD guideline 406

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

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The substance has been tested according to the OECD Guideline 406, Guinea pig Maximization Study before the LLNA test model was selected to be the preferred test system under the REACH Regulation.

Species:

guinea pig

Strain:

other: Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted)

Sex:

female

Details on test animals and environmental conditions:

Number of animals for main study / pretest: 15 female / 3 female animals (nulliparous and non-pregnant)
Age at delivery: 4 - 6 weeks
Age at pretest start/beginning of acclimatization period: 4 - 6 weeks
Body weight at pretest start: Pretest groups: 323 - 390 g
Body weight at beginning of acclimatization period: 301 - 360 g

No. of animals per dose:

The animals were distributed as follows:
1. Intradermal Pretest: 1 animal
2. Epidermal Pretest: 2 animals
3. Control Group: 5 animals
4. Test Group: 10 animals

Details on study design:

PEG 300 was used for the intradermal and epidermal pretests. It was also used for the intradermal and epidermal induction and the challenge in the main study. The 1:1 mixture (v/v) of Freund's Complete Adjuvant:physiological saline was used as vehicle for the pretest and the intradermal induction in the main study.

SELECTION OF CONCENTRATION OF TEST ITEM FOR MAIN STUDY:

Intradermal Induction:
The concentration of test item used for the intradermal induction exposure was well-tolerated systemically and was technically applicable concentration causing mild-to-moderate skin irritation.

Epidermal Induction:
The concentration of test item used for the epidermal induction exposure was well-tolerated and should have been the highest to cause mild-to-moderate skin irritation. In this study, no skin reactions were observed from the lowest to the highest tested concentration during the pretest.

Epidermal Challenge:
Concentration that was the maximum tested non-irritant concentration. To determine the different concentrations an intradermal and epidermal pretest was performed as described below.

STUDY CONDUCT - TREATMENT PROCEDURE

1. PRETEST PERFORMED WITH THE TEST SUBSTANCE BEFORE AND DURING THE ACCLIMATIZATION PERIOD OF THE CONTROL AND TEST GROUP

Intradermal injections:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of A = 5 %, B = 3 % and C = 1 % of the test item in PEG 300. The three concentrations were determined during formulation trials performed prior to the pretest. The concentration of 5 % was considered to be the highest technically applicable concentration which could be injected into the intra-cellular space in spite of the high viscosity of the application dilution and the obstacle caused by the tissues. Dermal reactions were assessed 24 hours later. Based on the results, the test item concentration of 5 % was selected for intradermal induction in the main study.

Epidermal applications:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (3 x 3 cm) were saturated with the test item at D = 50 % (technically the highest possible concentration to be applied sufficiently), E = 25 %, F = 15 % and G = 10 % in PEG 300 and applied to the clipped and shaved flanks. The amount of test item preparation applied was approximately 0.2 g for the test item at 50 % and a volume of approximately 0.2 ml was applied for the remaining test item concentrations. The patches were covered by a strip of aluminum foil and firmly secured by procedure ensured the intensive contact of the test item. The dressings were removed after an exposure period of 24 hours.
24 h after removal of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) in order to visualize any resulting erythema. The depilatory cream was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. Thereafter, the animals were dried with a disposable towel, and returned to their cages. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman. The allocation of the different test item dilutions to the sites on the two animals was alternated in order to minimize site-to-site variation in responsiveness.
Based on the results obtained the concentration selected for induction and challenge in the main study was 50 %.

2. MAIN STUDY

Intradermal injections / performed on test day 1
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) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline; 2) The test item, at 5 % in PEG 300; 3) The test item at 5 % in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Control Group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline; 2) PEG 300; 3) 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Epidermal applications / performed on test day 8
On test day 7 and 20 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) of the animals of the control and test group was clipped, shaved free of hair and the test area was pretreated with 0.5 ml of 10 % Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass rod without bandaging. This 10 % concentration of SLS enhances sensitization by provoking a mild inflammatory reaction (Magnusson and Kligman, 1970).
On test day 8, a 2 x 4 cm patch of filter paper was saturated with the test item (50 % in PEG 300) and placed over the injection sites of the test animals. The volume of test item preparation applied was approximately 0.3 ml. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test item.
The guinea pigs of the control group were treated as described above with PEG 300 only, also applied at a volume of approximately 0.3 ml. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

Challenge / performed on test day 22
The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way. Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 50 % (applied to the left flank) and the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The amount of test item preparation applied was approximately 0.2 g and a volume of approximately 0.2 ml was used for the vehicle. The dressings were left in place for 24 hours.
24 h after removal of the dressing the test sites treated with the test item were depilated as described in the epidermal pretest.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

INTERPRETATION
The results obtained from test animals following the challenge application were compared with the results seen in control animals.
An allergic reaction was defined by visible reddening of the challenge site.
If the dermal reactions of test animals following the 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 following the challenge were not clearly different from the reactions seen in the control group animals, the results for the test animals were considered "inconclusive".
The test animals were considered to show no evidence of contact hypersensitivity if the dermal reactions to the challenge application were identical or less marked and/or persistent than the reactions observed in the control animals.
By "maximizing" the exposure and enhancing allergenicity, some probles could arise, particularly in relation to specificity, especially the potential for false-positive reactions. An inflammatory response at challenge may not necessarily be due to allergenicity, but instead may be a fals-positive irritant response caused by an inducing hyperirritability.

Challenge controls:

see Details on Study design

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde

Positive control results:

CONTROL GROUP:
Discrete/patchy erythema was observed in all animals treated with PEG 300 only.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Test substance, 50 % in PEG 300 (left flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

PEG 300 only (right flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Test substance, 50 % in PEG 300 (left flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

PEG 300 only (right flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Test substance, 50 % in PEG 300 (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

PEG 300 only (right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Test substance, 50 % in PEG 300 (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

PEG 300 only (right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

1% in PEG 300

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

none

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

1% in PEG 300

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

none

Remarks on result:

positive indication of skin sensitisation

RESULTS - MAIN STUDY:

Skin effects after intradermal induction - Performed on test day 1

The expected and common findings were observed in the control and test group after the different applications using FCA intradermally. These findings consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation. No detailed description of the effects is given in the report as these FCA effects are well-known.

Skin effects after epidermal induction - Performed on test day 8

Control group: Discrete/patchy erythema was observed in all animals treated with PEG 300 only.

Test group: Discrete/patchy to moderate/confluent erythema were observed in all animals at the 24 -hour reading and discrete/patchy erythema at the 48 -hour reading after treatment with the test item at 50 % in PEG 300.

The reactions observed in both groups occurred following pretreatment with 10 % SLS in paraffinum perliquidum.

Skin effects after the challenge - performed on test day 22

Control group: No skin reactions were observed in the animals when treated with either PEG 300 only or when treated with the test item at 50 % in PEG 300.

Test group: No skin reactions were observed in the animals when treated with either PEG 300 only or when treated with the test item at 50 % in PEG 300.

Viability / Mortality / Macroscopic findings

There were no deaths during the course of the study, hence no necropsies were performed.

Clinical signs, systemic

No signs of systemic toxicity were observed in the animals.

Body weights

The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

other: not sensitizing

Conclusions:

Based on the above mentioned findings in a adjuvant sensitization test (M&K-test) in guinea pigs and in accordance to Commission Directive 96/54/EEC, the test substance has not to be classified and labelled as a skin sensitizer.

Executive summary:

In order to assess the cutaneous allergenic potential of the test substance, the Maximization-Test was performed in 15 (10 test and 5 control) female albino guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/EEC, B.6.

The intradermal induction of sensitization in the test group was performed in the nuchal region with a 5 % dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test item at 50 % in PEG 300 one week after the intradermal induction and following pretreatment of the test areas with 10 % Sodium-Lauryl-Sulfate (SLS) 20 hours prior to application of the test item. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion following pretreatment with 10 % SLS.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 50 % in PEG 300 and PEG 300 alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred. None of the control and test animals showed skin reactions after the challenge treatment with test substance at 50 % (w/w) in PEG.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A Maximization-Test was performed in 15 (10 test and 5 control) female albino guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/EEC, B.6.

The intradermal induction was performed with a 5 % dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted with the test item at 50 % in PEG 300 one week after the intradermal induction and following pretreatment of the test areas with 10 % Sodium-Lauryl-Sulfate (SLS) 20 hours prior to application of the test item. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion following pretreatment with 10 % SLS. None of the control and test animals showed skin reactions after the challenge treatment with test substance at 50 % (w/w) in PEG.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

As no sensitising response was observed in a reliable guinea pig study, no classification is required according to Regulation (EC) No 1272/2008 (CLP).