Fatty acids, C14-22, ethylene esters, bisulfited, sodium salts

Endpoint summary

Description of key information

Key study: Test method according to OECD 406. GLP Study. The subtance is not a skin sensitizer in the Guinea Pig Maximisation Test.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

Based on the available information, false positives were expected in the LLNA study with the test subtance.

Kreiling, R., H.M. Hollnagel, L. Hareng, D. Eigler, M.S. Lee, P. Griem, B. DreeBen, M. Kleber, A. Albrecht, C. Garcia, and A. Wendel (2008). Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT). Food and Chemical Toxicology. 46:1896-1904.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, 2040C
- Females, nulliparous and non-pregnant: yes
- Age at study initiation: 3 or 4 weeks old.
- Weight at study initiation: 274.3 ± 17 g(mean).
- Housing: In groups of three at the maximum in polycarbonate containers, the floowing of which was covered with dust-free cuttings and the top fitted with a stainless steel lid. with a feedinf device and drinking devide.
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Envigo, 2040C ad libitum.
- Acclimation period: 5 days
- Indication of any skin lesions: none.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22±2ºC
- Humidity (%): 30-70%
- Air changes (per hr): At least ten per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

olive oil

Concentration / amount:

2 % / 0.1 mL

Day(s)/duration:

Injection.

Adequacy of induction:

other: Maximal Non Necrotizing Concentration

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Remarks:

Sodium lauryl sulfate was used before the topical application of the test item

Concentration / amount:

100% / 0.5 mL

Day(s)/duration:

2 days

Adequacy of induction:

other: Maximal Non Irritant Concentration

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

100 % and 50 %

Day(s)/duration:

1 day

Adequacy of challenge:

other: Maximal Non Irritant Concentration

No. of animals per dose:

10 guinea pigs for the test item (treated)
5 guinea pigs as negative controls.

Details on study design:

RANGE FINDING TESTS:
Determination of the Maximal Non Nectrotizing Concentration (MNNC) by intradermal injection
This test was conducted for the purpose of defining a MNNC of the test item which, on intradermal injection during the induction phase, does not risk causing too great a lesion (non-necrotizing concentration), should be well-tolerated systemically and should be the highest to cause mild-tomoderate skin irritation.
Two animals received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 concentrations: diluted at 10%, 5%, 2% and 1% in olive oil in view to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after the injections.
Necrosis or slight necrosis were observed at the tested concentrations of 10% and 5%. 24 hours after the injections, no cutaneous reaction has been observed in the animals at the tested concentrations of 2% and 1%. The MNNC was 2 %.

Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC)
This test, which allowed evaluating the irritancy potential of the test item, defined whether an application of sodium lauryl sulfate would be needed during topical induction phase. The test item was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 hours, at 4 different concentrations: undiluted (100%) and diluted at 50%, 20% and 10% in liquid paraffin. After the removal of the occlusive dressing, the treated areas were rinsed with liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing.
24 hours after the removal of the occlusive dressings, no cutaneous reactions were noted whatever the tested concentration.
In view of these results, the concentration selected was 100% for the 2nd induction of the Group 2 and the MNIC determination began at the concentration of 100%.

Determination by topical application of the Maximal Non Irritant Concentration (MNIC) This test was carried out for the purpose of determining the MNIC of the test item without risk of an irritant effect during the challenge phase.
Three guinea pigs were treated according to the same treatment as animals from GROUP 1 (control) for the induction phase (i.e. olive oil and liquid paraffin). During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 hours at 4 different concentrations: undiluted (100%) and diluted at 50%, 20% and 10% in liquid paraffin. After the removal of the occlusive dressing, the treated areas were rinsed with distilled water and liquid paraffin.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressing.
24 and 48 hours after the removal of the occlusive dressings, no cutaneous reaction was noted whatever the tested concentration. In view of this result, the concentrations selected were 100% (MNIC) and 50% (1/2 MNIC).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: After 7 days of the application of the intradermal induction the topical induction was performed for 48 hours.
- Test groups:
Intradermal induction:
After shearing the scapular zone, three (3) pairs of intradermal injections (ID) of 0.1 mL were performed on the scapular zone in such a way as an injection on each pair is placed to either side of the spine as follows:

• 2 ID: Freund’s Complete Adjuvant diluted at 50 % in olive oil
• 2 ID: test item at 2% in olive oil
• 2 ID: a test mixture in equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and the test i tem at 4% in olive oil


After 7 days the scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulfate at 10% in thick vaseline, in order to create a local irritation.
One day later a topical application under occlusive dressing (25mm x 25mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for 48 hours was performed on the injection sites of each animal.

GROUP 1 (control): 0.5 mL of liquid paraffin. GROUP 2 (treated): 0.5 mL of the test item at 100%
- Duration: Not applicable for intradermal injection. 2 days for the topical induction.
- Concentrations: 2 % for the intradermal induction, 100 % for the topical one.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 1
- Exposure period: 24 hours
- Test and control groups:
The experimental procedure of this phase was identical for both groups GROUP 1 (Control) and GROUP 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application, under occlusive dressing, was performed during 24 hours: - 1 sample cup containing the test item undiluted 100% (MNIC) and 1 sample cup containing the test item diluted at 50% in liquid paraffin (1/2 MNIC).
- Evaluation (hr after challenge):
1st reading time – 24 hours after the patch removal.
2nd reading time – 48 hours after the patch removal.

Positive control substance(s):

yes

Remarks:

Hexylcinnamaldehyde (CAS 101-86-0)

Positive control results:

The results of the 3 last positive control groups (Reference substance: alpha-Hexylcinnamaldehyde) carried out in order to assess the sensitivity of the strain of guinea pig used at the laboratory to a known sensitiser were satisfactory. The reference substance α-Hexylcinnamaldehyde must be classified in category 1 “Skin sensitisati on” sub-category 1B. The signal word “Warning” and hazard statement H317 “ May cause an allergic skin reaction” are required.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Remarks:

CLP implementation-

Conclusions:

The test substance gave no indiciations of skin sensitisation in the Guinea Pig Maximisation Test.

Executive summary:

An in vivo study was conducted to determine the skin sensitisation properties of the test material according to the OECD guideline 406 under GLP conditions. The Guinea Pig Maximisation Test was used because false positives were predicted in the LLNA study (OECD guideline 429) based on the available literature and taking into account the structure of the test item. According to the preliminary studies, the main study consisted of an intradermal induction at 2 % and a topical aplication at 100 % 7 days later. After 10 days of rest phase, the challenge phase was performed under occlusive dressing for 24 hours and consisted of a single topical application of the test item at 100 % and diluted at 50 % in liquid paraffin. No macroscopic cutaneous reactions attributable to allergy were noted 24 or 48 hours after the patch removal. Based on the results it was concluded that the test item does not have to be classified in Category 1 as a skin sensitizer in accordance to CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

A Guinea Pig Maximisation Test was conducted since false positives were expected for the substance in the LLNA. Based on the results, the substance is not classified for skin sensitisation according to CLP (Regulation (EC) No 1272/2008) since there was no indication of skin sensitisation in any of the experimental animals.