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Toxicological information

Skin sensitisation

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
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)

Test material

Constituent 1
Test material form:
other: liquid
Details on test material:
Reference Name: H2925 (CAS No. 68130-53-0)Label Identification: HATCOL 2925, Lot No. 2013090401, 14 Mar 14Quantity & Date Received: 20 Mar 14; 679.5 g (GW)Physical Description: Colorless to light amber liquidStorage: Room temperaturePurity: Not provided to testing facilityStability: Not provided to testing facilityConcentrations Administered: 100%; 50 and 25% in 4:1 acetone:olive oilData generated for characterization and stability is the responsibility of the Sponsor.

In vivo test system

Test animals

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.

Study design: in vivo (LLNA)

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.

Results and discussion

Positive control results:
Reported in table form - detailed under Any other information

In vivo (LLNA)

Resultsopen allclose all
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

Any other information on results incl. tables

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

 

 

 

 

 

 

 

Applicant's summary and conclusion

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).