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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 406 and GLPs. The actual test method used was that of Kimber et al. (1989) and Basketter DA & Scholes EW (1992) as detailed in OECD Guideline 429.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 406 and GLPs. The actual test method used was that of Kimber et al. (1989) and Basketter DA & Scholes EW (1992) as detailed in OECD Guideline 429.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Reason / purpose:
read-across source
Positive control results:
Materials tested in the positive control studies included: α-hexylcinnamaldehyde,4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, Penicillin G sodium salt, polyoxyethylenesorbitan monooleate, eugenol, and cobalt chloride hexahydrate. All of these studies were positive in the LLNA when evaluated at the test laboratory.
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
0.5% w/v
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
5% w/v
Key result
Parameter:
SI
Value:
2.2
Test group / Remarks:
50% w/v
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle: 3829.14 0.5%: 5470.05 5%: 4899.16 50%: 8419.97

Disintegrations per minute/node:

Vehicle: 478.6

0.5%: 683.8

5%: 612.4

50%: 1052.5

 

Clinical observations:

No mortality or signs of toxicity were noted during the study. 

 

Body weights:

Body weights and body weight changes in test substance-treated animals were comparable to controls. 

Interpretation of results:
other: Not sensitising
Conclusions:
Under conditions of this study, Glycerol Ester of Tall Oil (resin acids and rosin acids, tall-oil, esters with glycerol) is not considered to be a skin sensitizer in female mice when tested in the Local Lymph Node Assay (LLNA) as described in OECD Guideline 429. Based on the absence of a threefold increase in the Stimulation Index at any of the tested concentrations, the test substance was classified as “not a moderate to strong sensitizer”. Based on an absence of positive effects in this study, Glycerol Ester of Tall Oil (resin acids and rosin acids, tall-oil, esters with glycerol) is not classifiable for Skin Sensitization according to UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. Glycerol Ester of Tall Oil (resin acids and rosin acids, tall-oil, esters with glycerol) is not classified for Skin Sensitization in the Annex I of Directive 67/548/EEC.
Executive summary:

This data is being read across from the source study that Resin acids and rosin acids, tall-oil, esters with glycerol based on category read across that is explained in the category justification document attached in Section 13 of the dossier.

In a skin sensitization study using the Local Lymph Node Assay, three groups of 4 (CBA/CaCruBR) female mice were administered daily applications of 50 µL (25 µL per ear) Glycerol Ester of Tall Oil at test concentrations of 0.5, 5, and 50% (w/v) in a 4:1 acetone:olive oil vehicle for 3 consecutive days.  A control group of 4 mice received the vehicle only. Five days after the first application, the animals were injected via the tail vein with 250 µL of phosphate buffered saline containing ^3H-methyl thymidine and euthanized 5 hours later. The auricular lymph nodes were collected and a single cell suspension was prepared for each dose group. The ^3HTdR incorporation by the cells was determined and a Stimulation Index (SI) calculated for each dose concentration of the test material. A Stimulation Index of less than 3.0 was recorded for all dose concentrations. Under the conditions of the study, Glycerol Ester of Tall Oil (resin acids and rosin acids, tall-oil, esters with glycerol) is not a moderate or strong skin sensitizer in mice.  

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
-Test material (as cited in report): Glycerol Ester of Tall Oil. Tall oil rosin is considered to be equivalent to Rosin. See justification for this provided in the CSR.
-Physical state: Pale yellow granular solid
-Date received: 2002-02-27
-Storage: approximately -18 °C in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Test animals:
-Source: Charles River UK Limited, Margate, Kent, UK
-Sex: Female
-Age at study initiation: 7-12 weeks
-Acclimation period: 5 days
-Weight at study initiation: 15-23 g
-Housing: Housed in groups of 4 in polypropylene cages fitted with stainless steel mesh lids and furnished with softwood woodflakes.
-Diet: Certified Rat and Mouse Diet (Code 5LF2; PMI Nutrition International, Nottingham, UK), ad libitum
-Environmental enrichment: Items were provided that would not impact the outcome of the study.
-Water: local municipality, ad libitum
-Method of animal identification: unique number that was written on the tail using a black indelible marker pen
-Method of animal distribution: animals were selected at random

Environmental Conditions:
-Temperature (°C): 19-25
-Humidity (%): 30-70
-Photoperiod: 12 hours light/12 hours dark
-Air exchanges: 15 per hour

In-Life Study Dates:
-Experimental Start Date: 2002-03-20
-Experimental Completion Date: 2002-03-26

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.5, 5, and 50%
No. of animals per dose:
4 females/group
Details on study design:
The test method used was that of Kimber et al. (1989) and Basketter DA & Scholes EW (1992) as detailed in OECD Guideline 429.

References:
Kimber I, Hilton J & Weisenberger C (1998). The Murine Local Lymph Node Assay for Identification of Contact Allergens: A Preliminary Evaluation of in situ Measurements of Lymphocyte Proliferation. Contact Dermatitis 21:215-20.

Basketter DA & Scholes EW (1992). Comparison of the Local Lymph Node Assay with the Guinea Pig Maximization Test for the Detection of a Range of Contact Allergens. Food and Chemical Toxicology 30:65-69.

Test material preparation:
The test substance was prepared in a glove bag under an inert atmosphere of nitrogen. High shear blending occurred using an ultratorax for a minimal period of 10 minutes and the temperature was kept below 60 °C. All preparations/dosing vessels were purged with nitrogen prior to removal from the glove bag. The test substance was freshly prepared in acetone/olive oil 4:1.

Verifications of concentration, homogeneity and stability of the test substance were not performed and were not requirements of the guidelines.

Test material administration:
Groups of 4 mice were treated with the test substance at concentrations of 0.5, 5, and 50% (w/v) in acetone/olive oil 4:1. The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days. The test substance was administered using a micropipette and spread over the dorsal surface of the ear using the tip of the pipette. An additional group of 4 mice received the vehicle alone in the same manner and served as study controls.

^3H-Methyl Thymidine administration:
Five days following the first topical application of the test substance, all mice were injected via the tail vein with 250 µL of phosphate buffered saline containing ^3H-methyl thymidine (^3HTdR:80 µCi/mL, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

Clinical observations:
All animals were observed on a daily basis. Any signs of toxicity or ill health were recorded during the study. Day 3 observations were not recorded but this was considered not to affect the integrity of the study.

Body weights:
The body weight of each animal was recorded on Day 0 (before dosing) and on Day 5 (prior to termination).

Termination:
Five hours following the administration of ^3HTdR, all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes were excised from each mouse and pooled together by group. One milliliter of phosphate buffered saline (PBS) solution was added to each of the pooled samples.

Preparation of single cell suspension:
A single cell suspension of the pooled lymph node cells was prepared by gentle mechanical disaggregation through 200-mesh stainless steel gauze. The cells were rinsed through the gauze with 4 mL of PBS into a petri dish. The lymph node cell suspension was transferred to a 10-mL centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove any remaining lymph node cells and the wash was added to the 10-mL centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 X g) for ten minutes, re-suspended in 10 mL of PBS, re-pelleted and re-suspended in 10 mL PBS. The radioactive material was precipitated by adding 3 mL of 5% trichloroacetic acid (TCA).

Determination of ^3HTdR incorporation:
After overnight incubation at 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 X g) for ten minutes, re-suspended in 1 mL of TCA, and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). The samples were placed in the scintillator for approximately 20 minutes in the dark to reduce the risk of luminescence. ^3HTdR incorporation was measured by β-scintillation counting using a Beckman LS6500 scintillation system. The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of ^3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (test/control ratio).

Interpretation of results:
The test substance was regarded as a sensitizer if at least one concentration of the test material resulted in a threefold or greater increase in ^3HTdR incorporation compared to control values. A test material that failed to produce a threefold or greater increase in ^3HTdR incorporation was classified as "not a moderate to strong sensitizer".
Positive control substance(s):
other: While not tested concurrently with this study, the sensitivity of the mouse strain was periodically checked by testing facility using several known positive controls and a table of results was provided in study report. Materials tested are listed below.
Statistics:
No data available

Results and discussion

Positive control results:
Materials tested in the positive control studies included: α-hexylcinnamaldehyde,4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, Penicillin G sodium salt, polyoxyethylenesorbitan monooleate, eugenol, and cobalt chloride hexahydrate. All of these studies were positive in the LLNA when evaluated at the test laboratory.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
0.5% w/v
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
5% w/v
Key result
Parameter:
SI
Value:
2.2
Test group / Remarks:
50% w/v
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle: 3829.14 0.5%: 5470.05 5%: 4899.16 50%: 8419.97

Any other information on results incl. tables

Disintegrations per minute/node:

Vehicle: 478.6

0.5%: 683.8

5%: 612.4

50%: 1052.5

 

Clinical observations:

No mortality or signs of toxicity were noted during the study. 

 

Body weights:

Body weights and body weight changes in test substance-treated animals were comparable to controls. 

Applicant's summary and conclusion

Interpretation of results:
other: Not sensitising
Conclusions:
Under conditions of this study, Glycerol Ester of Tall Oil (resin acids and rosin acids, tall-oil, esters with glycerol) is not considered to be a skin sensitizer in female mice when tested in the Local Lymph Node Assay (LLNA) as described in OECD Guideline 429. Based on the absence of a threefold increase in the Stimulation Index at any of the tested concentrations, the test substance was classified as “not a moderate to strong sensitizer”. Based on an absence of positive effects in this study, Glycerol Ester of Tall Oil (resin acids and rosin acids, tall-oil, esters with glycerol) is not classifiable for Skin Sensitization according to UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. Glycerol Ester of Tall Oil (resin acids and rosin acids, tall-oil, esters with glycerol) is not classified for Skin Sensitization in the Annex I of Directive 67/548/EEC.
Executive summary:

In a skin sensitization study using the Local Lymph Node Assay, three groups of 4 (CBA/CaCruBR) female mice were administered daily applications of 50 µL (25 µL per ear) Glycerol Ester of Tall Oil at test concentrations of 0.5, 5, and 50% (w/v) in a 4:1 acetone:olive oil vehicle for 3 consecutive days.  A control group of 4 mice received the vehicle only. Five days after the first application, the animals were injected via the tail vein with 250 µL of phosphate buffered saline containing ^3H-methyl thymidine and euthanized 5 hours later. The auricular lymph nodes were collected and a single cell suspension was prepared for each dose group. The ^3HTdR incorporation by the cells was determined and a Stimulation Index (SI) calculated for each dose concentration of the test material. A Stimulation Index of less than 3.0 was recorded for all dose concentrations. Under the conditions of the study, Glycerol Ester of Tall Oil (resin acids and rosin acids, tall-oil, esters with glycerol) is not a moderate or strong skin sensitizer in mice.