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Skin sensitisation

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Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March 2014 to 05 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guidelines in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species & Strain: Mouse; CBA/Jcr
Justification of Species: The mouse is species of choice for a local lymph node assay to provide information on which human hazard can be judged.
Source: Harlan Sprague-Dawley; Indianapolis, IN
Quantity & Sex: 5 females per each Test group & 5 females in each Control group (all nulliparous & non-pregnant)
Quarantine Period: 5 days
Date Born/Date Received: 21 Feb 14 / 17 Apr 14
Anima/Group Identification: Tail marking / Cage card
Weights on Initial Dose Day: 20.4-26.3 g

Cage Type: Polycarbonate boxes with bedding
Housing: 1 - 5 per cage
Environmental Controls: Set to Maintain: Temperature: 22° ± 3°C; Relative humidity: 30 - 70%; 12-hour light/dark cycle; 10+ air changes per hour
Temperature/Rel. Humidity: 19-23°C/52-84%
Food: PMI Feeds Inc.™ Formulab #5008; available ad libitum
Water: Municipal water supply analyzed by TCEQ Water Utilities Division; available ad libitum from automatic water system
Animal husbandry and housing at STILLMEADOW, Inc. comply with standards outlined in "Guide for the Care and Use of Laboratory Animals" (NRC Publ.). No contaminants were expected to have been present in feed or water that would have interfered with or affected results of the study.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Each Test animal in its group received an open application of 25 µL of appropriate dilution (25 or 50%) of test substance, or 100% test substance undiluted.
No. of animals per dose:
5 mice per dose group
Details on study design:
Test Substance Preparation and Administration: Healthy mice were released from quarantine prior to testing. Five females were selected for each of three Test groups (Groups I- III). On Days 1, 2 and 3, each Test animal in its group received an open application of 25 µL of appropriate dilution (25 or 50%) of test substance, or 100% test substance undiluted, to the dorsum of both ears. The Vehicle Control group (5 females) was treated the same way as test animals, but with vehicle alone instead of test substance. The Positive Control group (5 females) was treated with alpha-hexylcinnamaldehyde as received. All Test and Control animals were given a two-day rest period on Days 4 and 5.

Injection of Tritiated Methvl-Thymidine: On Day 6 of the study, all Test and Control animals were injected in the tail vein with 250 µL of 0.01 M phosphate-buffered saline (PBS; Sigma, Lot 081M8207, Exp Nov 2021), pH 7.4 at 25°C per manufacturer, containing 20 µCi of [methyl-3H) Thymidine (PerkinElmer, Lot 201404, Exp Apr 2015). Five hours after injection, animals were sacrificed with an overdose of CO2, the draining auricular lymph nodes excised and pairs from each individual animal processed.

Suspension Preparation and DPM Determination: A single cell suspension was prepared by gentle mechanical disintegration through 200 mesh stainless steel gauze. Cells were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA; Ricca Chemical, Lot 2403 813, Exp Mar 20 15) at 4°C for 18 hours. The pellets were resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. Incorporation of tritiated thymidine was measured by liquid scintillation counting as disintegrations per minute (DPM) from paired lymph nodes of each animal, and mean DPM/animal was calculated for each group.

Body Weights and Observations: Individual body weights were recorded on Day 1 prior to dosing, and Day 6, prior to injection. All Test and Control animals were observed daily for clinical signs of toxicity and any signs of excessive irritation at the test site.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not specified in the study report.
Positive control results:
Reported in table form - detailed under Any other information
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
Test Group I
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
Test Group II
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
Test Group III

Stimulation Index or Test/Vehicle Control Ration derived for each Test group based on group mean DPM is as follows.

Animal Group

Test Substance Concentration

Average Count per Mouse

Number if Mice in Group

Test/Vehicle Control Ratio

Vehicle Control

NA

699

5

NA

Test Group I

25%

1173

5

1.7

Test Group II

50%

993

5

1.4

Test Group III

100%

917

5

1.3

Positive Control

NA

7696

5

11.0*

NA – Not applicable; *-Positive Control used to confirm animal sensitization potential and validate procedures.

 

TABLE 1 – Body Weights and DPM Counts

Test Substance: H2925 (CAS No. 68130-53-0)

Skin Sensitization: Local Lymph Node Assay in Mice

Animal No. (in Group)

Day of Study

DPM Count

Day 1 Wts.

Day 6 Wts.

Vehicle Control Group

1

23.5

24.3

967

2

24.2

25.0

1143

3

23.4

23.7

751

4

23.3

23.8

355

5

22.9

23.2

279

Test Group I – 25% concentration

1

26.3

27.3

811

2

24.8

24.9

664

3

21.5

22.6

1254

4

22.5

23.3

1461

5

25.4

25.9

1676

Test Group II – 50% concentration

1

25.2

25.6

1963

2

22.6

24.1

814

3

20.4

21.1

628

4

23.0

23.5

732

5

24.0

24.3

827

Test Group III – 100% concentration

1

24.1

24.4

836

2

23.2

24.0

615

3

24.9

25.1

525

4

22.1

23.4

673

5

24.5

25.2

1935

Positive Control Group

1

22.2

22.4

6743

2

22.7

23.4

1542

3

23.7

23.8

859

4

22.3

22.1

20841

5

23.8

23.9

8493

Note: Body weights are in grams.

 

TABLE 2 – Observations of Clinical Signs

Test Substance: H2925 (CAS No. 68130-53-0)

Skin Sensitization: Local Lymph Node Assay in Mice

Vehicle Control

 

Day

Reaction and Severity

1

2

3

4

5

6

Appeared normal at each observation

 

 

 

 

 

 

Test Group I – 25% concentration

 

Day

Reaction and Severity

1

2

3

4

5

6

Appeared normal at each observation

 

 

 

 

 

 

Test Group II – 50% concentration

 

Day

Reaction and Severity

1

2

3

4

5

6

Appeared normal at each observation

 

 

 

 

 

 

Test Group III – 100% concentration

 

Day

Reaction and Severity

1

2

3

4

5

6

Appeared normal at each observation

 

 

 

 

 

 

Positive Control

 

Day

Reaction and Severity

1

2

3

4

5

6

Appeared normal at each observation

 

 

 

 

 

 

 

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
H2925 (CAS No. 68130-53-0) produced a stimulation index < 3 in all groups of Test animals, and is not therefore considered a sensitizer (defined as producing a positive response).
Executive summary:

A skin sensitization study was conducted on 3 groups of 5 female mice to determine if test substance H2925 (CAS No. 68130-53-0) possesses a significant potential to cause skin sensitization. Five females were assigned to each of three groups, designated Groups I -III. Test groups were treated with an appropriate dilution (25 or 50%) in 4: 1 acetone: olive oil, or 100% test substance. Each animal received 25 µltothe dorsum of each ear. Animals were treated once daily for three days. After a two-day rest period, all animals were injected with tritiated methyl-thymidine in the tail vein. Five hours later, animals were sacrificed, and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A Vehicle Control group of five females was run concurrently, treated in the same manner with vehicle only instead of test substance or dilution. A Positive Control group of five females was also run concurrently, treated with alpha-hexylcinnamaldehyde as received.

The test substance produced a stimulation index < 3 in all groups of Test animals, and is not therefore considered a sensitizer (defined as producing a positive response).

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Oct 1996 - 21 Jan 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Magnussen and Kligmann Method
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Available supporting data is >12 years old. LLNA is available as a key study.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HRP inc. and Charles River Laboratories, USA
- Age at study initiation: 5-7 weeks
- Weight at study initiation: 391- 489 g (males); 366 - 480 g (females)
- Housing: Individually housed in suspended, stainless steel cages with wire mesh bottoms
- Diet: Agway Prolab Purina Guinea Pig Diet; ad libitum
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature and humidity was controlled daily
- Photoperiod (hrs dark / hrs light): 12 / 12 h
Route:
intradermal and epicutaneous
Vehicle:
propylene glycol
Concentration / amount:
Induction: Intradermal injection 5%, epicutaneous application 100%
Challenge: 100%
Route:
epicutaneous, open
Vehicle:
propylene glycol
Concentration / amount:
Induction: Intradermal injection 5%, epicutaneous application 100%
Challenge: 100%
No. of animals per dose:
20
Details on study design:
RANGE FINDING TESTS: Yes (Intradermal: October 1st 1996 - October 3rd 1996; Topical: October 1st 1996 - October 4th 1996)
Two animals were pre-treated with two intradermal injections of FCA/water emulsion (1:1) approx. one week prior to test material administration.
Animals were administered 0.1 mL of the test substance (5% w/v) in propylene glycol via injection on either side of the spinal column and observed for dermal irritation 24 h and 48 h after injection.
For the topical range-finding study animals (3 per sex) were dosed with 0.1 mL of four different concentrations (25%, 50%, 75% and 100% in ethyl alcohol) at different sites, two on either side of the column. Treatment was for 24 h under occlusive conditions and observed for dermal irritation 24 h and 48 h afterwards.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Test groups: 1
- Control group: 2 (positive and irritation control)
- Site: A row of three injections was made on each side in the shoulder region.
- Frequency of applications: Days 1 and 8
- Duration: 25 days
- Concentrations: Intradermal induction: 5%, Topical induction: 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 14 d after last induction application
- Exposure period: 24 h
- Test groups: 1
- Control group: 2 (positive and irritation control)
- Site: Flank
- Concentrations: 100%
- Evaluation (hr after challenge): 24 and 48 h after removal of the patches
Positive control substance(s):
yes
Remarks:
Hexylcinnamic aldehyde (HCA)
Positive control results:
Treatment with 50% HCA resulted in 30% positive response.
Treatment with 100% HCA resulted in 20% positive response (not sensitising, by definition). This lower effect might be due to decreased dermal absorption at high substance concentration.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
100%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 100%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 50%. No with. + reactions: 3.0. Total no. in groups: 10.0.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 1.0. Total no. in groups: 20.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
100%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 100%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 50%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
CLP: not classified
DSD: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a LLNA conducted in accordance with OECD Guideline 429, the test substance produced a stimulation index < 3 in all groups of test animals, therefore the test substance is not considered a be a skin sensitiser.

In addition, a guinea pig maximisation test was conducted with read-across substance, decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS No. 11138-60-6) in accordance with OECD Guideline 406. A total of 20 male and female Dunkin-Harley guinea pigs were treated with the test substance and compared with 10 negative control animals. The sensitivity of the animal strain was tested using Hexylcinnamic aldehyde (HCA) as positive control substance. A 5% dilution of the test substance in propylene glycol was used for intradermal induction and 100% used for epidermal induction of the scapular region on days 1 and 8 .Fourteen days after the last induction treatment, all animals were challenged epicutaneously with the undiluted test substance. Twenty-four hours after challenge one out of twenty animals showed a skin reaction compared to two out of ten for the positive control. Forty-eight hours after challenge exposure all skin examination scores were zero in all test animals. Under the conditions of this test, the read-across substance was not classified as a sensitiser.

Migrated from Short description of key information:

The substance is not classified as a skin sensitiser (OECD Guideline 429).

Justification for selection of skin sensitisation endpoint:

Klimisch 1 study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the above mentioned results, classification according to the CLP Regulation (EC)1272/2008 is not required.