Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1981, page 57 ff.

Deviations:

yes

Remarks:

no pretreatment with sodium lauryl sulphate)

Principles of method if other than guideline:

Test concentration used for induction was the highest non-irritating concentration and not the lowest concentration which causes mild-to-moderate skin irritation. The animals were not pretreated with sodium lauryl sulphate, as required by recent guidelines for testing of non-irritating substances. Furthermore, no positive control substance was tested in parallel and no periodically performed sensitivity and reliability check of the experimental technique is reported.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The sulfosuccinate substances might give false positive reactions in the LLNA test.

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite

EC Number:

939-654-5

Molecular formula:

Molecular formula cannot be given as the substance is a mixture

IUPAC Name:

Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite

Test material form:

other: liquid

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Pirbright / Hoe: DHPK(SPF-LAC.)/Boe

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Lippische Versuchtierzucht Hagemann GmbH & Co. KG , Hameiner Strasse 3, 4923 Exertal 1
- Age at study initiation: Not provided
- Weight at study initiation: 228-293 g
- Fasting period before study: Not provided
- Housing: Max. 5 guinea pigs/cage, Makrolon IV cages, (h 20 x b 30x l 55 cm)
- Diet (e.g. ad libitum): Ad libitum, Ssniff -G® (Alleindiät für Meerschweinchen), Ssniff Spezialdiäten GmbH, 4770 Soest/Westfalen, pellets, 1.0 cm long, 0.5 cm diameter
- Water (e.g. ad libitum): Ad libitum, Makrolon bottles (Firma Becker & Co., 4620 Castrop-Rauxel), tap water as for human use, analytical and bacteriological controls every half year
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 2 °C (measured with thermohygrometer twice daily)
- Humidity (%): 50-85% (measured with thermohygrometer twice daily)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12 (12hours daylight from 7.00-19.00h)

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: deionised water

Induction 2

Concentration / amount:

No information on content of active ingredient of the test substance in this study report. However, according to producer information substance [Trade name] has 40 % act. ingr.
INDUCTION:
- intradermal treatment: 10 % test substance (corresponding to 4 % act.ingr.)
- epicutaneous treatment: 50 % test substance (corresponding to 20 % act.ingr.)

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: deionised water

Challenge 2

Concentration / amount:

No information on content of active ingredient of the test substance in this study report. However, according to producer information substance [Trade name] has 40 % act. ingr.
CHALLENGE: 50 % test substance (corresponding to 20 % act.ingr.)

No. of animals per dose:

20 (10 male, 10 female)

Details on study design:

RANGE FINDING TESTS: In order to exclude primary skin irritation, two guinea pigs underwent single dermal treatment under occlusive conditions with the following concentrations (0.5 mL / animal): 100% (= undiluted), 75%, 50% and 10% solution in deionised water (corresponding to 40, 30, 20 and 4% act.ingr., respectively). An area of approx. 8x5 cm over the schoulders was clipped short. Two animals per concentration were treated.

MAIN STUDY
A. INDUCTION EXPOSURE first stage (intradermal)
- No. of exposures: 6 sites in 40 animals
- Exposure period: single injection
- Test group: 10 male and 10 female guinea pigs (1 group with 3 bilateral injections)
- Control group: 10 male and 10 female guinea pigs (1 group with 3 bilateral injections)
- Site: bilaterally to the spine starting in the cranio-dorsal area
- Frequency of applications: 1 (6 sites)
- Concentrations: - Test groups: 0.05 mL injection of 10% solution of the test substance in deionised water (2 injection sites bilaterally to the spine); 0.05 mL injection of 10% solution of the test substance in Freund Complete Adjuvans (2 injection sites bilaterally to the spine); 0.05 mL injection of Freund Complete Adjuvans undiluted (2 injection sites bilaterally to the spine)
- Control group: 0.05 mL injection of Freund Complete Adjuvans undiluted (2 injection sites bilaterally to the spine); 0.05 mL injection of 10% deionised water in Freund Complete Adjuvans (2 injection sites bilaterally to the spine); 0.05 mL injection of undiluted deionised water (2 injection sites bilaterally to the spine)

B.INDUCTION EXPOSURE second stage (epicutaneous)
- No. of exposures: 2 (x 6 sites) in 40 animals
- Day(s) of challenge: 7 days
- Exposure period: 48 hours occlusive dressing
- Test group: 20 animals
- Control group: 20 animals
- Site: the 3 sites bilaterally of the spine
- Concentrations: - Test group: 0.5mL of a 50% solution of the test substance in deionised water (corresponding to 20% act.ingr.)
- Control group: 0.5 mL of undiluted deionised water


C. CHALLENGE EXPOSURE 1
- No. of exposures: 2 (x 6 sites) in 40 animals
- Day(s) of challenge: 3 weeks (after intradermal injection)= 21 days
- Exposure period: 24 hours
- Test group: 20 animals
- Control group: 20 animals
- Site: the 3 sites bilaterally of the spine
- Concentrations: - Test group: 0.5mL of a 50% solution of the test substance in deionised water on the left side; 0.5 mL of undiluted deionised water on the right side
- Control group: 0.5mL of a 50% solution of the test substance in deionised water on the left side; 0.5 mL of undiluted deionised water on the right side

- Evaluation (hr after challenge): 24 h and 48h

Challenge controls:

yes: 0.5 mL of undiluted deionised water on the right side

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50% test substance (corresponding to 20% act.ingr.)

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

one animal showed a moderate erythema (reaction score 2)

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 % test substance (corresonding to 20% act.ingr.)

No. with + reactions:

0

Total no. in group:

20

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 % test substance (corresponding to 20% act.ingr.)

No. with + reactions:

0

Total no. in group:

20

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50% test substance (corresponding to 20% act.ingr.)

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

one animal showed a slight erythema (reaction score 1)

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

No. with + reactions:

0

Total no. in group:

0

Remarks on result:

other: no positive control group

Any other information on results incl. tables

Range finding:100%: 24h and 48h : slightly erythema; 75%: 24h : very slightly erythema; 50% and 10%: no primary dermal irritation

Main test:
Test group: 24h after retesting (50% solution): 1 animal with moderate erythema. 48h after retesting all 20 animals of the test group had no reaction.
Control group: after 24h no reaction on test site or on control site. At 48 h 1/20 animals showed a slight erythema (score 1) at the test substance treated skin area.
19 of the 20 test animals had no reaction, thus the test substance [Trade name] (50% solution) can be classified as “probably not sensitizing”. The significant irritation in one test animal can still be assigned to chance.

Table 1. Range finding: individual observations

Animal number	Sex	Concentration	24h	48h
1	m	100% undiluted	1	0
2	f	100% undiluted	1	±
1	m	75% in deionised water	±	0
2	f	75% in deionised water	0	0
1	m	50% in deionised water	0	0
2	f	50% in deionised water	0	0
1	m	10% in deionised water	0	0

 

Table2. Individual observations after retesting (Attempt C) in the Test group (Right flank: 0.5 mL deionised water; Left flank: 0.5 mL test substance 50% solution in deionised water)

Animal number	Sex	24 hours		48 hours
		Right flank	Left flank	Right flank	Left flank
1	M	0	0	0	0
2	M	0	0	0	0
3	M	0	0	0	0
4	M	0	0	0	0
5	M	0	2	0	0
6	M	0	0	0	0
7	M	0	0	0	0
8	M	0	0	0	0
9	M	0	0	0	0
10	M	0	0	0	0
11	F	0	0	0	0
12	F	0	0	0	0
13	F	0	0	0	0
14	F	0	0	0	0
15	F	0	0	0	0
16	F	0	0	0	0
17	F	0	0	0	0
18	F	0	0	0	0
19	F	0	0	0	0
20	F	0	0	0	0

 

Table3. Individual observations after retesting (Attempt C) in the Control group (Right flank: 0.5 mL deionised water; Left flank: 0.5 mL test substance 50% solution in deionised water)

Animal number	Sex	24 hours		48 hours
		Right flank	Left flank	Right flank	Left flank
1	M	0	0	0	0
2	M	0	0	0	0
3	M	0	0	0	0
4	M	0	0	0	0
5	M	0	0	0	1
6	M	0	0	0	0
7	M	0	0	0	0
8	M	0	0	0	0
9	M	0	0	0	0
10	M	0	0	0	0
11	F	0	0	0	0
12	F	0	0	0	0
13	F	0	0	0	0
14	F	0	0	0	0
15	F	0	0	0	0
16	F	0	0	0	0
17	F	0	0	0	0
18	F	0	0	0	0
19	F	0	0	0	0
20	F	0	0	0	0

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

Based on current study, the test item can be classified as “probably not sensitising” as 19 of the 20 test animals had no reaction to the test substance (50% solution) according to the method of Magnusson and Kligman (OECD 406) and the distinct irritation in 1 test animal can still be assigned to chance.

Executive summary:

After an acclimation period of at least 7 days, 2 groups (test and control group) of 20 animals each were tested. Before treatment the animals were shaved in the shoulder area (approx. 8 x 5 cm). In order to exclude primary dermal irritation, 2 guinea pigs were tested in a single dermal (occlusive) application (0.5 mL (g) /animal) with following concentrations: 100% undiluted, 75%, 50% and 10% solution in deionised water of a test formulation containing 40% active ingredient (=range finding test).

In Attempt A (intradermal induction) each animal was injected in 2 injection sites (pairs) bilaterally to the spine (starting in the cranio-dorsal area) with 0.05 mL as follows:

Test group:1 pair (2) injections of test substance 10% solution in deionised water; 1 pair (2) injections of test substance 10% solution in FCA and 1 pair (2) injections of FCA undiluted.

Control group:1 pair (2) injections of FCA undiluted; 1 pair (2) injections of dionised water 10% solution in FCA and 1 pair (2) injections of deionised water undiluted.

In Attempt B , 7 days after intradermal induction, the first dermal induction started with 0.5 mL of the test substance (50% solution= maximal concentration with no primary irritation in range finding) on the left flank and 0.5 mL vehicle (deionised water) on the right flank in a closed patch test. After 48 hours the dressing was removed.

In Attempt C (Challenge), 3 weeks after intradermal treatment, the second single dermal application or challenge was executed with “Hill-Top” –Chambers, with 0.5 mL of the test substance (50% solution) on the left flank and 0.5 mL vehicle (deionised water) on the right flank. After 24 hours the dressing was removed. 24 Hours and 48 hours after removal, evaluation was done.

The range finding showed no primary irritation in the 50% and 10% solution group.

 In Attempt C (Challenge) there was 1 animal in the test group (50% solution) with slightly erythema at 24h evaluation. At 48h after challenge none of the 20 animals of the test group had any reaction. In the control group after 24 and 48h there were no reactions on the test site or the control site. In 19 of the 20 test animals no reaction was observed, thus the test substance (50% solution or 20% active ingredient) can be classified as “probably not sensitizing”. The significant irritation in 1 test animal can still be assigned to chance.