Soybean oil, maleated, ester with triethanolamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

Modified Buehler Method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11mApr 2017 Experimental starting date (Primary Irritation Phase – animals shaved) to 01 Jun 2017 Experimental termination (completion) date (scheduled euthanasia)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

EPA OPPTS Guideline 870.2600 OECD Guideline 406
EEC Guideline 92/69, Annex V, B6

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

The substance is surface active and can form micelles. Surface active materials have been demonstrated to induce false positive responses in the LLNA but not in standard guinea-pig assays. The Buehler method is more humane than the Magnusson-Kligman maximisation test, and therefore it was selected for use to evaluate this substance instead of the LLNA.

Test material

Test material information

Constituent 1

Reference substance name:

Soybean oil, maleated, ester with triethanolamine

EC Number:

285-107-2

EC Name:

Soybean oil, maleated, ester with triethanolamine

Cas Number:

85029-82-9

Molecular formula:

C63H113NO12 to C67H123NO12

IUPAC Name:

Soybean oil, maleated, ester with triethanolamine

Test material form:

other: Clear amber viscous liquid

Details on test material:

100% product
Physical state/Appearance: Clear amber viscous liquid
Expiry Date: 24 June 2018
Storage Conditions: Room temperature in the dark

Specific details on test material used for the study:

Exp. date: 17 Feb 2019
[ID No. 170071]
Brown, translucent, viscous liquid
Date of Receipt 23 Feb 2017

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Remarks:

Hartley (Crl:HA) albino guinea pigs

Sex:

male/female

Details on test animals and environmental conditions:

Hartley (Crl:HA) albino guinea pigs were used as the test system on this study. The animal model, the Hartley albino guinea pig, is generally recognized as appropriate for skin sensitization studies. The number of animals selected for this study (4 animals/sex for the Primary Irritation Group, 10 animals/sex/group for the Test Group, 5 animals/sex for the Naïve Control-I Group, and 5 animals/sex for the Positive Control Group) was the number required to satisfy regulatory guidelines.
The albino guinea pigs utilized for this study were received in good health from Charles River Laboratories, Inc., Stone Ridge, NY. The guinea pigs were inspected by a qualified technician upon receipt, weighed, and uniquely identified by a cage card displaying the animal number.
The guinea pigs were acclimated to laboratory conditions for a minimum of 5 days. During the acclimation period, the guinea pigs were observed twice daily for mortality and moribundity.

All animals were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 68 °F to 78°F (20°C to 26°C) and 30% to 70%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. These data are summarized in Appendix 3. Actual mean daily temperature ranged from 72.5°F to 73.3°F (22.5°C to 22.9°C) and mean daily relative humidity ranged from 44.6% to 52.9% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. Lighting conditions were recorded every 15 minutes. The 12-hour light/12-hour dark photoperiod was interrupted if necessary to allow for the performance of protocol-specified activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour

Upon arrival, all animals were housed in individual suspended wire-mesh cages. The animals were maintained by the animal husbandry staff of Charles River in accordance with SOPs.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals.2 The animal facilities at Charles River Ashland are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

The basal diet used in this study, PMI Nutrition International, LLC, Certified Guinea Pig LabDiet® 5026, is a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River. Municipal water supplying the facility was analyzed for contaminants according to Charles River SOPs. The results of the diet and water analyses are maintained at Charles River. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. The basal diet and municipal water, delivered by an automatic watering system, were provided ad libitum throughout the acclimation period and during the study.


Animals used on the Primary Irritation Phase were arbitrarily selected from available stock. Animals used in the main study were selected from available stock and assigned to groups by stratified randomization through use of WTDMS™. The selected animals were young adult; body weight values ranged from 473 g to 655 g for males and from 398 g to 554 g for females at randomization excluding Primary Irritation Phase animals (see Appendix 1 - Study Protocol and Deviations).

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Remarks:

100% Test Substance

Concentration / amount:

100% Test Substance

Day(s)/duration:

3 induction doses spaced 1 week apart over a period of 3 weeks

Adequacy of induction:

highest technically applicable concentration used

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

The Challenge Phase consisted of a single application of the maximal nonirritating concentration of test substance to determine if delayed contact hypersensitivity had occurred.

Day(s)/duration:

48 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

4 animals / sex for the Primary Irritation Group, 10 animals / sex / group for the Test Group, 5 animals / sex for the Naïve Control-I Group, and 5 animals / sex for the Positive Control Group

Details on study design:

The positive control substance was α-hexylcinnamaldehyde (HCA) (Lot No. MKBT2800V, exp. date: 25 Nov 2020).
Acetone, NF (Lot No. 2EL0104, exp. date: 03 Dec 2020) and polyethylene glycol (PEG) 400, NF (Lot No. 2EI0410, retest date: 19 Jun 2018) were used to prepare the HCA formulations for the Challenge and Rechallenge Phases.

Primary Irritation Phase
The test substance was prepared for dosing the Primary Irritation Phase as weight-to-volume (w/v) mixtures in propylene glycol (Lot No. 165702, retest date: 30 Sep 2019) at concentrations of 1%, 2.5%, 5%, 10%, 25%, 50%, 75%, and 100%. The appropriate amount of test substance for each concentration was weighed and vehicle was added in sufficient quantity to obtain the desired concentrations. Sufficient undiluted test substance was also dispensed for dosing.

Induction Phase
Sufficient amounts of undiluted test substance and undiluted positive control substance were dispensed for induction dosing.

Challenge Phase
A sufficient amount of undiluted test substance was dispensed for the Test Group and Naïve Control-I Group.

Challenge controls:

The Challenge Phase consisted of a single application of the maximal nonirritating concentration of test substance to determine if delayed contact hypersensitivity had occurred.
Two weeks after the final induction dose, undiluted test substance was administered to previously unexposed sites on the posterior left flank of the Test and Naïve Control-I Group

animals at 0.3 mL/site. For the Positive Control Group, 10% and 20% concentrations of HCA in 70% acetone/30% PEG 400 were administered to previously unexposed sites on the posterior left and right flanks, respectively, at 0.3 mL/site. Doses were applied under 25-mm Hill Top Chambers® that were occluded with plastic wrap and overwrapped with nonirritating elastic tape. All challenge exposures were approximately 6 hours, after which the bandages were removed and the sites wiped with disposable paper towels moistened with propylene glycol (see
Appendix 1 - Study Protocol and Deviations). On the following day, at least 3 hours prior to the 24-hour scoring, the hair was carefully clipped with an electric shaver from the dosing area of each animal.
One week after the initial challenge, a rechallenge was performed in which the positive control substance (10% and 20% concentrations of HCA in 70% acetone/30% PEG 400) was administered to previously unexposed sites on the middle right and left flanks, respectively, at
0.3 mL/site (see Appendix 1 - Study Protocol and Deviations). Doses were applied under 25-mm Hill Top Chambers® that were occluded with plastic wrap and overwrapped with
nonirritating elastic tape. All rechallenge exposures were approximately 6 hours, after which the bandages were removed and the sites wiped with disposable paper towels moistened with propylene glycol (see attached Appendix 1 - Study Protocol and Deviations).

Positive control substance(s):

yes

Remarks:

The positive control substance was α-hexylcinnamaldehyde Acetone, NF & polyethylene glycol, retest date: 19 Jun 2018) were used to prepare the HCA formulations for the Challenge and Rechallenge Phases.

Results and discussion

Positive control results:

The concentration used at challenge for the Positive Control Group was a known non-irritating concentration; therefore, grades of 1 or greater in the Positive Control Group indicated sensitization responses.
The concurrent Positive Control Group exhibited an incidence index of 0% following the challenge exposure. The response requirement for a non-adjuvant positive control group is at least 15%. As a result, a rechallenge was conducted for the Positive Control Group. Following rechallenge, the positive control article, HCA, was a sensitizer when administered as both a 10% concentration (20% incidence index) and 20% concentration (80% incidence index), supporting the sensitivity and reliability of the experimental design

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

There were no clinical findings observed during the study.

Remarks on result:

no indication of skin sensitisation

Remarks:

Challenge Dosing

Results 2

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

There were no clinical findings observed during the study.

Remarks on result:

no indication of skin sensitisation

Remarks:

Challenge Dosing

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

10% HCA

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

There were no clinical findings observed during the study.

Remarks on result:

other: See remarks

Remarks:

The concurrent Positive Control Group exhibited an incidence index of 0% following the challenge exposure. The response requirement for a non-adjuvant positive control group is at least 15%. As a result, a rechallenge was conducted for the Positive Control Group. Following rechallenge, the positive control article, HCA, was a sensitizer when administered as both a 10% concentration (20% incidence index) and 20% concentration (80% incidence index), supporting the sensitivity and reliability of the experimental design

Results 4

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

positive control

Dose level:

10% HCA

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

There were no clinical findings observed during the study.

Remarks on result:

other: See remarks

Remarks:

The concurrent Positive Control Group exhibited an incidence index of 0% following the challenge exposure. The response requirement for a non-adjuvant positive control group is at least 15%. As a result, a rechallenge was conducted for the Positive Control Group. Following rechallenge, the positive control article, HCA, was a sensitizer when administered as both a 10% concentration (20% incidence index) and 20% concentration (80% incidence index), supporting the sensitivity and reliability of the experimental design

Results 5

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

20% HCA

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

There were no clinical findings observed during the study.

Remarks on result:

other: See remarks

Remarks:

The concurrent Positive Control Group exhibited an incidence index of 0% following the challenge exposure. The response requirement for a non-adjuvant positive control group is at least 15%. As a result, a rechallenge was conducted for the Positive Control Group. Following rechallenge, the positive control article, HCA, was a sensitizer when administered as both a 10% concentration (20% incidence index) and 20% concentration (80% incidence index), supporting the sensitivity and reliability of the experimental design

Results 6

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

positive control

Dose level:

20% HCA

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

There were no clinical findings observed during the study.

Remarks on result:

other: See remarks

Remarks:

The concurrent Positive Control Group exhibited an incidence index of 0% following the challenge exposure. The response requirement for a non-adjuvant positive control group is at least 15%. As a result, a rechallenge was conducted for the Positive Control Group. Following rechallenge, the positive control article, HCA, was a sensitizer when administered as both a 10% concentration (20% incidence index) and 20% concentration (80% incidence index), supporting the sensitivity and reliability of the experimental design

Results 7

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

positive control

Dose level:

10% HCA

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

There were no clinical findings observed during the study.

Remarks on result:

positive indication of skin sensitisation

Remarks:

The concurrent Positive Control Group exhibited an incidence index of 0% following the challenge exposure. The response requirement for a non-adjuvant positive control group is at least 15%. As a result, a rechallenge was conducted for the Positive Control Group. Following rechallenge, the positive control article, HCA, was a sensitizer when administered as both a 10% concentration (20% incidence index) and 20% concentration (80% incidence index), supporting the sensitivity and reliability of the experimental design

Results 8

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

positive control

Dose level:

10% HCA

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

There were no clinical findings observed during the study.

Remarks on result:

positive indication of skin sensitisation

Remarks:

The concurrent Positive Control Group exhibited an incidence index of 0% following the challenge exposure. The response requirement for a non-adjuvant positive control group is at least 15%. As a result, a rechallenge was conducted for the Positive Control Group. Following rechallenge, the positive control article, HCA, was a sensitizer when administered as both a 10% concentration (20% incidence index) and 20% concentration (80% incidence index), supporting the sensitivity and reliability of the experimental design

Results 9

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

positive control

Dose level:

20% HCA

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

There were no clinical findings observed during the study.

Remarks on result:

positive indication of skin sensitisation

Remarks:

The concurrent Positive Control Group exhibited an incidence index of 0% following the challenge exposure. The response requirement for a non-adjuvant positive control group is at least 15%. As a result, a rechallenge was conducted for the Positive Control Group. Following rechallenge, the positive control article, HCA, was a sensitizer when administered as both a 10% concentration (20% incidence index) and 20% concentration (80% incidence index), supporting the sensitivity and reliability of the experimental design

Results 10

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

positive control

Dose level:

20% HCA

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

There were no clinical findings observed during the study.

Remarks on result:

positive indication of skin sensitisation

Remarks:

The concurrent Positive Control Group exhibited an incidence index of 0% following the challenge exposure. The response requirement for a non-adjuvant positive control group is at least 15%. As a result, a rechallenge was conducted for the Positive Control Group. Following rechallenge, the positive control article, HCA, was a sensitizer when administered as both a 10% concentration (20% incidence index) and 20% concentration (80% incidence index), supporting the sensitivity and reliability of the experimental design

Any other information on results incl. tables

There were no deaths during the study.

There were no clinical findings observed during the study.

There were no remarkable body weight changes noted during the study.

Dermal Observations and Severity Indices

Data: see attached Table 3, Table 6, Table 7, Table 8, Table 9, Table 10

Text Table 5 Challenge Dosing

Group	Material	Dermal Scores										Severity Index		Incidence Index
		24-hour					48-hour					24-hour	48-hour
		0	±	1	2	3	0	±	1	2	3
Test	100 % OS200034M	19	1	0	0	0	19	1	0	0	0	0.0	0.0	0%
NaïveControl-I	100 % OS200034M	10	0	0	0	0	10	0	0	0	0	0.0	0.0	NA
Positive Control	10% HCA	7	3	0	0	0	9	1	0	0	0	0.2	0.1	0%
Positive Control	20% HCA	3	7	0	0	0	7	3	0	0	0	0.4	0.2	0%

 

 

Text Table 6 Rechallenge Dosing

Group	Material	Dermal Scores										Severity Index		Incidence Index
		24-hour					48-hour					24-hour	48-hour
		0	±	1	2	3	0	±	1	2	3
Positive Control	10% HCA	3	5	2	0	0	4	6	0	0	0	0.5	0.3	20%
Positive Control	20% HCA	0	5	5	0	0	4	3	3	0	0	0.8	0.5	80%

 

Based on the severity and incidence indices for the Test Group, there was no indication of a sensitization response following the challenge dosing. Therefore, a rechallenge was not warranted for the Test Group.

The concurrent Positive Control Group exhibited an incidence index of 0% following the challenge exposure. The response requirement for a non-adjuvant positive control group is at least 15%. As a result, a rechallenge was conducted for the Positive Control Group. Following rechallenge, the positive control article, HCA, was a sensitizer when administered as both a 10% concentration (20% incidence index) and 20% concentration (80% incidence index), supporting the sensitivity and reliability of the experimental design.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Under the conditions of this study the test item was a non-sensitizer in albino guinea pigs.

Conclusions:

Under the conditions of this study the test item was a non-sensitizer in albino guinea pigs.

Executive summary:

Objective

The objective of this study was to determine the ability of the test item to induce delayed contact hypersensitivity when applied in close contact to the skin of albino guinea pigs.

Study Design

The sensitization potential of the test item was evaluated in this modified Buehler method dermal sensitization study.

A Test Group of 10 male and 10 female Hartley (Crl:HA) albino guinea pigs was dosed topically with the test substance 1 time per week for 3 weeks for a total of 3 induction exposures. The duration of each exposure was 6 hours. Approximately 2 weeks after the last induction exposure, the animals were challenge-dosed for detection of sensitization by topical application of the test substance to a previously unexposed area of skin. The test substance, was administered at a concentration of 100% (Induction and Challenge).

A Naïve Control-I Group of 5 male and 5 female guinea pigs was dosed with the test substance in the challenge phase in the same manner as the Test Group and served as an irritation control.

A Positive Control Group of 5 male and 5 female guinea pigs was included to demonstrate the reliability of the experimental design. The Positive Control Group was induced and challenged on a similar regimen as the Test Group. The positive control material, α-hexylcinnamaldehyde (HCA), was administered at a concentration of 100% (Induction) and at concentrations of

10% and 20% (Challenge) prepared v/v in 70% acetone/30% PEG 400. Seven days after challenge dosing, Positive Control Group animals were rechallenged to support the initial challenge results.

Reactions to challenge and rechallenge dosing were evaluated at approximately 24 and 48 hours after completion of exposure. Body weights and clinical observations were recorded at randomization (Study Day -1) and at study termination (Study Day 34 or 37).

Prior to conducting the induction and challenge phases of the study, 4 male and 4 female guinea pigs were dosed to determine the appropriate concentrations of test substance to be used for dosing.

 

Results

There were no deaths, test substance-related clinical findings, or remarkable body weight changes during the study period. The Incidence Index for the Test Group was 0% (0/20) following challenge dosing.

 

Conclusions

Under the conditions of this study, the test item was a non-sensitizer in albino guinea pigs.