Amines, di-C16-18-alkyl

Endpoint summary

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:

08-Jun-2010 to 16-Jul-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This is the scientific justification for performing the OECD Test No. 406 for Skin Sensitisation, i.e. the Guinea Pig Maximization Test (GPMT), instead of1he required OECD Test No. 429, i.e. Skin Senitisation according to the Local Lymph Node Assay.

In recently published articles in peer reviewed journals. see reference list, it is clearly demonstrated that surfactants are more likely to give rise to false positives in the LLNA. Consequently, in the evaluation of such substances for sensitizing properties the LLNA test is not an appropriate assay and would not represent an optimum use of test animals. It is therefore recommended that the Guinea Pig Maximization Test (GPMT) is used instead. This is also supported by the TG OECD 406 ”In addition, test substance classes or substances containing functional groups shown to act as potential confounders (Basketter et al., 2009) may necessitate
the use of guinea pig tests".
References:
Kreiling. R .. Hollnagel, H..M ; Hareng. L.. Eigler D. .. Lee. M.S . Griem. P .. Dreesen B,
Klebcr. M ., Albrecht. A.. Garcia. C .• Wendel, A (2008) ,Comparison of the skin
sensitizing potential of unsaturated compounds and assessed by the murine
local lymph node assay (LLNA) and the guinca pig maximization test (GPMT).
Food Chem. Toxicol. 46. 1896 – 1904

D. Basketter. N. Ball. S. Cagen,. JC Carrillo. H. Certa. D. Eigler. H. Esch. C. Garcia, C. Graham. C.Haux. R.
Kreiling. A. Mehling. (2009) Application of a weight of evidence approach to assessing discordant sensitisation
datasets: Jmplications for REACH. Reg Tox Pharrn. 55:90-96.

C. Garcia. . N. Ball. S. Cagen,. JC Carrillo. H. Certa. D. Eigler. H. Esch C. Graham.. C.Haux, R. Kreiling. A.
Mehling. (2010) Comparative testing for the identification of skin sensitizing potentials of nonionic sugar lipid
surfactants. Reg Tox Pharrn 58: 301-307
Note: Ref list not complete due to lack of space.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / The Netherlands
- Number of Animals for Pretest / Main Test: 5 males / 15 males
- Animals of either sex are acceptable for use according to guidelines Commission Regulation (EC) No 440/2008, B.6 and OECD 406.
- Age at Pretest Start / Beginning of Acclimatization Period: 4-5 weeks
- Body Weight at Pretest Start: Pretest groups: 303 - 396 g
- Body Weight at Beginning of Acclimatization Period: Test and control groups: 352 - 415 g
- Identification: By unique cage number and corresponding individual ear tag.
- Randomization: Selected by hand at time of delivery. No computer generated randomization program.
- Acclimatization: Thirteen days for the test and control groups of the main test under standard laboratory conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used for the study. A certificate of health was provided by the animal supplier at animal delivery and included in the raw data.
- Accommodation: Individually in Makrolon type-4 cages with standard softwood bedding (‘Lignocel’ J. Rettenmaier&Söhne GmbH&CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland).
- Diet: Pelleted standard Provimi Kliba 3418 guinea pig breeding / maintenance diet batch no. 09/10, containing Vitamin C (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland), ad libitum. A haystick was also provided for environmental enrichment. Results of analyses for contaminants are archived aHarlan Laboratories Ltd.
Water: Community tap water from Füllinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at Harlan Laboratories Ltd.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 30-70%
- Air changes: 10 - 15 air changes per hour
- Photoperiod: 12 hours light and 12 hours dark

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Remarks:

Identification: Corn oil Description: Yellowish oily liquid Batch Number: 049103168 Source: Carl Roth GmbH & Co, 76185 Karlsruhe / Germany Expiry Date: 31-Jan-2014 Storage Conditions: At room temperature (range of 20 ± 5 °C), light protected.

Concentration / amount:

Intradermal induction: 1% (w/w) in corn oil
Epidermal induction: 50% (w/w) in corn oil
Epidermal challenge: 0.1% (w/w) in corn oil

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Remarks:

Identification: Corn oil Description: Yellowish oily liquid Batch Number: 049103168 Source: Carl Roth GmbH & Co, 76185 Karlsruhe / Germany Expiry Date: 31-Jan-2014 Storage Conditions: At room temperature (range of 20 ± 5 °C), light protected.

Concentration / amount:

Intradermal induction: 1% (w/w) in corn oil
Epidermal induction: 50% (w/w) in corn oil
Epidermal challenge: 0.1% (w/w) in corn oil

No. of animals per dose:

5 control and 10 test for the main test
(1 animal for the intradermal pretest and 4 animals for the epidermal pretest)

Details on study design:

Induction
1) Intradermal Injection / 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 just within the boundaries 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 1% in corn oil.
3) The test item at 1% 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) corn oil.
3) 1:1 (w/w) mixture of corn oil in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

2) Epidermal Induction / Performed on Test Day 8

One week after the intradermal injections, the scapular area (approximately 6 x 8 cm) was again shaved prior to epidermal induction. A 2 x 4 cm patch of filter paper was saturated with the test item at 50% in corn oil and placed over the injection sites of the test animals. The amount of test item preparation applied was approximately 0.3 g. 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 corn oil only, 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.


TREATMENT METHOD
The animal's fur was shaved with a fine clipper blade just prior to exposure. Intradermal injections or closed patches were applied to the animals as follows:

0.1 mL/site for the intradermal administrations or 0.2 to 0.3 g (or mL) for the epidermal administrations on a patch of filter paper.

The patch was covered by a strip of aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressing was left in place for 24 (epidermal pretest and epidermal challenge) or 48 hours (epidermal induction).
Identical patching method was used for the epidermal pretest, epidermal induction and epidermal challenge.

Challenge controls:

3) Challenge / Performed on Test Day 22
The test and control guinea pigs were challenged two weeks after epidermal induction and treated in the same way.
Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 0.1% (applied to the left flank) and the vehicle only (corn oil applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation applied was approximately 0.2 mL and a volume of approximately 0.2 mL was used for the vehicle. The dressings were left in place for 24 hours

Positive control substance(s):

yes

Remarks:

ALPHA-HEXYLCINNAMALDEHYDE

Positive control results:

The study was performed with 15 (10 test and 5 control) male albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF, delivered by Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / The Netherlands.

The intradermal induction of sensitization in the test group was performed in the nuchal region with a 10% 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 10% in PEG 300 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 3% in PEG 300 and PEG 300 alone under occlusive dressing.

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

Skin Effects in the Challenge:
Control Group:
No skin reactions were observed in the animals when treated with PEG 300 alone or when treated with the test item at 3% in PEG 300.

Test Group:
No skin reactions were observed in the animals when treated with PEG 300 alone.
Discrete/patchy erythema was observed in seven animals (70%) at the 24- and 48-hour readings after treated with the test item at 3% in PEG 300. Scaling was observed in one animal at the 48-hour reading.

No toxic signs were evident in the guinea pigs of the control or test group.

No deaths occurred.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Amines, bis(hydrogenated tallow alkyl) (CAS number: 61789-79-5), 0.1% in corn oil (left flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Not reported

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Amines, bis(hydrogenated tallow alkyl) (CAS number: 61789-79-5), 0.1% in corn oil (left flank) . No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Not reported.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Corn oil right flank

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Not reported

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Corn oil right flank. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Not reported.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Amines, bis(hydrogenated tallow alkyl) (CAS number: 61789-79-5), 0.1% in corn oil (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Not reported

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Amines, bis(hydrogenated tallow alkyl) (CAS number: 61789-79-5), 0.1% in corn oil (left flank) . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Not reported.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Corn oil right flank

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Not reported

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Corn oil right flank. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Not reported.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Amines, bis(hydrogenated tallow alkyl) (CAS number: 61789-79-5), 0.1% in corn oil (left flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Not reported

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Amines, bis(hydrogenated tallow alkyl) (CAS number: 61789-79-5), 0.1% in corn oil (left flank) . No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Not reported.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Corn oil right flank

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Not reported

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Corn oil right flank. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Not reported.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Amines, bis(hydrogenated tallow alkyl) (CAS number: 61789-79-5), 0.1% in corn oil (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Not reported

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Amines, bis(hydrogenated tallow alkyl) (CAS number: 61789-79-5), 0.1% in corn oil (left flank) . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Not reported.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Corn oil right flank

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Not reported

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Corn oil right flank. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Not reported.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Based on the above mentioned findings in an adjuvant sensitisation test (M&K-test) in guinea pigs and in accordance to Regulation (EC) No 1272/2008, Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) does not have to be classified and labeled as a skin sensitizer.

Executive summary:

In order to assess the cutaneous allergenic potential of Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4), the Maximization-Test was performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance with OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6.

 

The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 1% dilution of the test item in corn oil and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the test item at 50% in corn oil one week after the intradermal induction. The animals of the control group were intradermally induced with corn oil and FCA/physiological saline and epidermally induced with corn oil under occlusion.

 

Two weeks after epidermal induction the test and control animals were challenged by epidermal application of the test item at 0.1% in corn oil and corn oil alone under occlusive dressing.

 

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

 

Results - Skin Reactions after the Challenge Procedure

 

	After 24 hours				After 48 hours
	Positive / Total				Positive / Total
	% Positive of Total				% Positive of Total
Control Group
Amines, bis(hydrogenated tallow alkyl) (CAS		0 / 5				0 / 5
number: 61789-79-5), 0.1% in corn oil (left flank)		0				0
Corn oil only		0 / 5				0 / 5
(right flank)		0				0
Test Group
Amines, bis(hydrogenated tallow alkyl) (CAS		0 / 10				0 / 10
number: 61789-79-5), 0.1% in corn oil (left flank)		0				0
Corn oil only		0 / 10				0 / 10
(right flank)		0				0

No necropsy was performed on all surviving animals.

The control and test animals of the main test and the second epidermal pretest were sacrificed at the end of the observation period by intraperitoneal injection of pentobarbitone at a dose of 2.0 mL/kg of 162 mg sodium pentobarbitone/mL and discarded. The intradermal and epidermal I pretest animals were sacrificed as described above at the treatment start of the main test.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The potential of the test item Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman OECD Guideline 406 and EC 96/54/EEC, B.6 guideline. The study was conducted in compliance with GLP.

Based on preliminary testing, the induction by intradermal injection with the test item was performed at the concentration of 1% in corn oil (treated group) or vehicle alone (control group), the test item at the concentration of 1% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl (50/50, v/v) (control group). On day 8 the topical induction was performed when the animals of the treated group received the test item at the concentration of 50% (w/w) in corn oil to the same test site, which was then covered by an occlusive dressing for 48 hours. The challenge was performed on day 22 when 0.1% of the substance in corn oil was applied on the treated group.

No systemic clinical signs and no deaths were noted during the study. No relevant cutaneous reactions were observed at 24 and 48 hours after the challenge application. Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4), does not induce delayed contact hypersensitivity in guinea pigs.

Migrated from Short description of key information:

The potential of the test item Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406, 17th July 1992) and EC (96/54/EEC, B.6, 30th July 1996) guidelines and in compliance with Good Laboratory Principles. In the study with reliability rating 1, Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4)  did not induce delayed contact hypersensitivity in guinea pigs.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There are no guidelines for an animal test for respiratory sensitization, however in general respiratory sensitizers are also skin sensitizers. Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) was not found to be a skin sensitizer in the OECD 406 GLP study. This indicates that the substance is unlikely to posses any significant potential for respiratory sensitization.  Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) is a solid substance with a low vapour pressure of 2 mPa at 20°C and therefore inhalation exposure is unlikely.

Migrated from Short description of key information:

Data on respiratory sensitization on Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) is lacking, but since it is a solid substance with a low vapour pressure of 2 mPa at 20°C inhalation exposure is unlikely.

Justification for classification or non-classification

Skin

Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) was not found to be a skin sensitizer when tested in the OECD 406 GLP study.

Inhalation
Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) was not found to be a skin sensitizer in the OECD 406 GLP study. This indicates that the substance is unlikely to posses any significant potential for respiratory sensitization.  Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) is a solid substance with a low vapour pressure of 2 mPa at 20°C and therefore inhalation exposure is unlikely. Data on acute inhalation is lacking, but taken the result from the skin sensitization study and the low potential for inhalation exposure into consideration, the substance it is not classified as a respiratory sensitizer.