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Description of key information

Weight-of-evidence on sensitisation potential was available from registered substance and following read across substances in a guinea pig maximisation model and human patch test, all indicating there is no sensitisation potential:

-'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl)) amino]ethyl]esters, disodium salts'

-'Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts'.

Based on the negative findings in both models for different similar substances, potential for skin sensitisation can be excluded. 

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:
weight of evidence
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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.
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)

Route:
intradermal and epicutaneous
Vehicle:
other: deionised water
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.)

Route:
epicutaneous, occlusive
Vehicle:
other: deionised water
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
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)
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
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
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)
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

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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Test data were available for the registered substance, however read-across data were also used from N2 and N3 subgroup members as weight-of-evidence:

- 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts'

- 'Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts'.

Justification for read across within the category of N-containing sulphosuccinates (N2 and N3 subcategories) is documented in a separate document attached in Section 13.

 

Guinea-pig maximization

- In a skin sensitisation study with the registered substance according to the method of Magnusson and Kligman, 2 groups (test and control group) of 20 animals each were tested with the registered substance containing 40% active ingredient (Sterner and Chibanguza, 1988). Intradermal induction was done in 10 male and 10 female control and test animals with 0.05 mL. Test group:1 pair (2) injections of test substance 10% solution (4% active ingredient) 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 deionised water 10% solution in FCA and 1 pair (2) injections of deionised water undiluted. Epicutaneous induction was done, 7 days after intradermal induction; the first dermal treatment started with 0.5 mL of the test substance (50% solution= 20% active ingredient) 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. Challenge, 3 weeks after intradermal treatment, was done with 0.5 mL of the test substance (50% solution = 20% active ingredient ) on the left flank and 0.5 mL vehicle (deionised water) on the right flank. After challenge with 50% solution (20% active ingredient), there was 1 animal in the test group 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 sensitising”. The significant irritation in 1 test animal can still be assigned to chance.

- In a skin sensitisation study with read-across substance 'Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts' according to the method of Magnusson and Kligman in guinea pigs, the test item containing 39.80% active ingredient was tested for its sensitising properties (Haferkorn, 2013d).A 0.5% suspension in aqua ad iniectabilia chosen for the 1st (intracutaneous) induction stage revealed a discrete or patchy erythema in all 10 animals 24 and 48 hours after administration. 2 mL of a 25% suspension of the test item in aqua ad iniectabilia/animal chosen for the 2nd (topical) induction stage revealed a moderate and confluent erythema 48 hours and a discrete or patchy erythema 72 hours after start of exposure in all 10 animals. The challenge with 2 mL of a 10% suspension of the test item in aqua ad iniectabilia /animal - the maximum non-irritating concentration - revealed no skin irritation in any animal and, thus, the test item had no sensitising properties. The vehicle control revealed no skin reactions.

Animals of the same strain treated with α-hexyl cinnamic aldehyde in sesame oil exhibited a sensitising reaction in all animals in form of a moderate and confluent erythema (grade 2).Behaviour of the animals remained unchanged. Body weights were not influenced. The test item containing 39.80% active ingredient, was found to be not sensitising.

 

Human patch testing

In a skin sensitisation study with read across substance 'Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts', a 15 mm patch of the test item containing 35.8% active ingredient (2.5% in petrolatum) was applied to patch sites on the backs or volar forearms of 100 subjects for ten alternate-day 24 hour periods under occlusion (Kligman, 1977). Following a seven-day rest period, 15mm challenge patches of the test item (1% in petrolatum) were applied in the same manner to fresh sites on the backs or volar forearms of all 100 subjects for 24 hours. Challenge sites were read on removal of the patch and 24 hours thereafter, using the 0-4 scale. There were no instances of irritation or sensitisation from this material on the Draize-Shelanski Patch Test. It is unlikely that this test item would present a danger of irritation or sensitisation in normal, intended use.

 

Conclusion

- Based on the negative findings in the guinea-pig maximisation and human patch testing, potential for skin sensitisation of registered substance can be excluded.

- Further information supporting the absence of sensitisation potential is also provided in the read across justification for the N2 & N3 subgroups, showing that other guinea-pig maximisation was also negative (justification with data matrix separately attached in Section 13).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on these results and according to the CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for sensitisation.

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