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Description of key information

Skin sensitisation (LLNA): The test item is not considered a sensitizer (defined as producing a positive response). 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jan 18 to 21 Feb 18.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Relative humidity at times outside protocol range; one animal weight slightly over + 20% mean per protocol. The deviation(s) did not affect outcome of the study.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
No further details specified in the study report.
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species & Strain: Mouse; CBA/JcrHSD
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: The Jackson Laboratory; Bar Harbor, ME
Quantity & Sex: 5 females/each Test group & 5 females/each Control group
Acclimation Period: At least 5 days
Date Born/Date Received: 12 Dec 17 / 06 Feb 18
Animal Identification: Tail marking & cage card
Weights on Day 1: 18.8 - 25.8 g
Animal Husbandry
Housing & Cage Type: 1 - 5/cage in polycarbonate box & bedding; PVC pipe provided as enrichment
Environmental Controls
Set to Maintain:
∙ 21 + 3°C target temperature
∙ 30 - 70% target humidity
∙ 12-hr light/12-hr dark cycle
∙ 10+ air changes per hour
Actual Temp/Humidity: 20 - 24°C / 36 - 82%
Food: Teklad Global Diets® #2018; 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 study results.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100%; 50 and 25% in 4:1 v/v acetone:olive oil
No. of animals per dose:
5 females/each Test group & 5 females/each Control group
Details on study design:
Test Item 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 itemin 4:1 v/v acetone:olive oil vehicle, or 100% test item, 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 item. The Positive Control group (5 females) was treated with 60% alpha-hexylcinnamaldehyde in vehicle. All Test and Control animals were given a two-day rest period (Days 4 - 5).

Injection of Tritiated Methyl-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 SLBS4223, Exp May 2028), pH 7.4 at 25oC per manufacturer, containing 20 μCi of [methyl-3H] Thymidine (PerkinElmer, Lot 201801, Exp Jan 2019). Five hours after injection, animals were sacrificed by CO2 overdose, 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, Lot 1706G56, Exp Jun 2018; Lot 4710D97, Exp Sep 2018) at 4oC 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 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 excessive test site erythema.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A one-way parametric analysis of variance (ANOVA) with Dunnett’s Multiple Comparisons Test, using GraphPad InStat for Windows, GraphPad Software, San Diego California USA, was performed on DPM counts and body weights. As the SI was < 3 in all Test groups, an EC3 (used to classify a sensitizing test item) was not calculated.
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
100%
Key result
Parameter:
SI
Value:
9.1
Test group / Remarks:
Positive Control Group
Cellular proliferation data / Observations:
One each of Test Groups I and II, and Positive Control animals lost weight during the study. For the Day 6 body weights, the Test Group I mean body weight was statistically significantly greater than the control group (p=0.0135). There was no statistical difference in the Day 1 mean body weights against control (p>0.05 per ANOVA). In-life signs included matted fur in all groups; dose-site erythema in all groups except Vehicle; hunched posture and thinness in Positive controls only.

 Stimulation (SI) or Test/Vehicle Control Ratio derived for each Test group based on group mean DPM is as follows:

Animal Group

Test item Concentration

Average Count per Mouse

Number of Mice in Group

Test/Vehicle Control Ratio

Vehicle Control

NA

1575

4*

NA

Test Group I

25%

1175

5

0.8

Test Group II

50%

1312

5

0.8

Test Group III

100%

1749

5

1.1

Positive Control

NA

14355

5

9.1**

NA – Not applicable; *-Low count for one group animal considered procedure error & not used in calculations; ** - Positive Control used to confirm animal sensitization potential and validate procedures.

 

Body Weights and DPM Counts

Test Item: Petroleum Resins (Kendex 0897)

Skin Sensitization: Local Lymph Node Assay in Mice

 

Animal No. (in Group)

Day of Study

DPM Count

Day 1 Wt.

Day 6 Wt.

Vehicle Control Group

1

22.4

22.5

*

2

23.3

23.7

1010

3

21.0

22.4

1364

4

18.8

19.5

2622

5

19.9

20.8

1305

Mean

21.1

21.8

1575

Standard Deviation

1.8

1.6

715

Test Group I – 25% Concentration

1

20.8

21.7

1321

2

21.4

23.6

903

3

25.8

25.2

1432

4

24.0

24.4

976

5

21.6

24.5

1244

Mean

22.7

23.9

1175

Standard Deviation

2.1

1.3

227

Test Group II – 50% Concentration

1

22.2

22.0

1204

2

21.2

21.9

1358

3

20.8

22.3

947

4

20.3

21.6

1797

5

21.6

22.2

1256

Mean

21.1

22.0

1312

Standard Deviation

0.7

0.3

310

Test Group III – 100% Concentration

1

21.8

22.1

1260

2

22.2

22.4

1250

3

21.7

22.3

1951

4

21.8

22.2

2074

5

21.7

22.2

2209

Mean

21.8

22.2

1749

Standard Deviation

0.2

0.1

460

Positive Control Group

1

21.0

22.6

9490

2

20.5

21.4

17814

3

20.8

22.1

18001

4

19.3

20.0

10487

5

19.8

19.4

15984

Mean

20.3

21.1

14355

Standard Deviation

0.7

1.4

4079

*-Low number (89) considered procedure error, not used in calculations

Note: Body weights are in grams

 

Observations of Clinical Signs

Test Item: Petroleum Resins (Kendex 0897)

Skin Sensitization: Local Lymph Node Assay in Mice

Vehicle Control

Day

Reaction and Severity

1

2

3

4

5

6

Ear fur matted

5

5

5

5

0

0

Test Group I – 25% concentration

Day

Reaction and Severity

1

2

3

4

5

6

Ear fur matted

Dose-site erythema (v)

5

5

5

5

5

5

5

2

1

0

0

0

Test Group II – 50% concentration

Day

Reaction and Severity

1

2

3

4

5

6

Ear fur matted

Dose-site erythema (v-s)

5

5

5

5

5

5

5

2

5

1

2

0

Test Group III – 100% concentration

Day

Reaction and Severity

1

2

3

4

5

6

Ear fur matted

Dose-site erythema (v-s)

5

5

5

5

5

5

5

5

5

3

5

1

Positive Control

Day

Reaction and Severity

1

2

3

4

5

6

Ear fur matted

Dose-site erythema (v-s)

Thinness & hunched posture

5

0

0

5

5

0

5

5

0

5

5

2

5

5

2

2

3

1

v – very slight; s – slight/well-defined; m – moderate; e – extreme/severe

Note: Digits indicate number of animals exhibiting reaction.

Interpretation of results:
GHS criteria not met
Conclusions:
Petroleum Resins (Kendex 0897) 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 item Petroleum Resins (Kendex 0897) 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 vehicle, or 100% test item. Each animal received 25 μL to the 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 item or dilution. A Positive Control group of five females was also run concurrently, treated with 60% alpha-hexylcinnamaldehyde in vehicle.

 

The test item 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 conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A skin sensitization study was conducted on 3 groups of 5 female mice to determine if test item Petroleum Resins (Kendex 0897) 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 vehicle, or 100% test item. Each animal received 25 μL to the 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 item or dilution. A Positive Control group of five females was also run concurrently, treated with 60% alpha-hexylcinnamaldehyde in vehicle.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test item produced a stimulation index < 3 in all groups of Test animals, and is not therefore considered a sensitizer (defined as producing a positive response) and is Not Classified as a sensitiser in accordance with the CLP Regulation.