Tall oil, maleated

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, maleated

EC Number:

268-859-6

EC Name:

Tall oil, maleated

Cas Number:

68152-93-2

Molecular formula:

UVCB

IUPAC Name:

3,7-dimethyl-14,16-dioxo-19-(propan-2-yl)-15-oxapentacyclo[10.5.2.0²,¹¹.0³,⁸.0¹³,¹⁷]nonadeca-13(17),18-diene-7-carboxylic acid; 8-(7-hexyl-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)octanoic acid

Test material form:

liquid

Details on test material:

Lot No.: HD0258QH13

Specific details on test material used for the study:

Viscous brown liquid, Batch: HD0379UD11

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Source: Janvier, Le Genest-Saint-Isle, France
Age at the Initiation of Dosing: Young adult animals (approximately 11 weeks old) were selected.
Weight at the Initiation of Dosing: 19.4 to 24.7 g.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0%, 0.5%, 1%, 2 %

No. of animals per dose:

4 groups (0%, 0.5%, 1%, 2%) of 5 animals/group

Details on study design:

Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. A Certificate of Analysis or equivalent document was provided to the Test Facility.

Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.
Any residual volumes were discarded.

Justification for Test System and Number of Animals
The CBA/J mouse was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHW). The test method and number of animals were based on the test guidelines.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ±20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
The disposition of all animals was documented in the study records.

Husbandry
Housing
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The room(s) in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Environmental Conditions
Target temperatures of 18 to 24 °C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 21 to 22 °C with an actual daily mean relative humidity of 42 to 46%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Experimental Design
Justification of Route and Dose Levels
Dose route and dose concentrations used are in compliance with the OECD test guidelines for LLNA studies.

Positive control substance(s):

other: For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks showing reproducible and consistent positive results.

Statistics:

All results are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥3, the test item may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments.
The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation

Results and discussion

Positive control results:

n/a

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Value:

0.9

Results 2

Parameter:

SI

Value:

2.5

Test group / Remarks:

0.5%

Results 3

Parameter:

SI

Value:

3.2

Test group / Remarks:

1%

Results 4

Parameter:

SI

Value:

8.1

Test group / Remarks:

2%

Any other information on results incl. tables

Scaliness was noted on one ear of one animal treated at 2% on Day 4, which was considered not to have a toxicologically significant effect on the activity of the nodes.  

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

All auricular lymph nodes of the animals of the test item treated groups were considered enlarged, auricular lymph nodes of the control group were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 0.5, 1 and 2% were 706, 891 and 2256 DPM, respectively. The mean DPM/animal value for the vehicle control group was 277 DPM. The SI values calculated for the test item concentrations 0.5, 1 and 2% were 2.5, 3.2 and 8.1, respectively.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

The results indicate that the test item could elicit a SI ≥3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI = 3) of 0.9% was calculated.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Based on these results:
• according to the recommendations made in the test guidelines (including all amendments), EnvaMul 600 would be regarded as skin sensitizer.
• according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), EnvaMul 600 should be classified as skin sensitizer (Category 1A).
• according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), EnvaMul 600 should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.

Executive summary:

A skin sensitisation study was performed on the test item EnvaMul 600.

The study was carried out based on the guidelines described in:

·        OECD, Section 4, Health Effects, No.429 (2010),

·        EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"

·        EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

 

Test item concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 0.5, 1 or 2% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Aceton:Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with ³H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

 

Scaliness was noted on one ear of one animal treated at 2% on Day 4, which was considered not to have a toxicologically significant effect on the activity of the nodes. 

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

All auricular lymph nodes of the animals of the test item treated groups were considered enlarged, auricular lymph nodes of the control group were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 0.5, 1 and 2% were 706, 891 and 2256 DPM, respectively. The mean DPM/animal value for the vehicle control group was 277 DPM. The SI values calculated for the test item concentrations 0.5, 1 and 2% were 2.5, 3.2 and 8.1, respectively.

These results indicate that the test item could elicit a SI ≥3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI = 3) of 0.9% was calculated.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results:

·        according to the recommendations made in the test guidelines (including all amendments), EnvaMul 600 would be regarded as skin sensitizer.

·        according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), EnvaMul 600 should be classified as skin sensitizer (Category 1A).

·        according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), EnvaMul 600 should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.