Tall oil, maleated

Endpoint summary

Description of key information

An IN VITRO skin sensitisation study has been performed on test item EnvaMul 600 according to OECD guideline 442d. The test item is classified as positive in the KeratinoSens^TM assay since positive results (>1.5-fold induction) were observed at test concentrations of <200 µg/mL with a cell viability of >70% compared to the vehicle control in two out of three experiments.

An IN VIVO study has been 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”.

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.

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

 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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)

Specific details on test material used for the study:

Viscous brown liquid, Batch: HD0379UD11

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.

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

Positive control results:

n/a

Parameter:

EC3

Value:

0.9

Parameter:

SI

Value:

2.5

Test group / Remarks:

0.5%

Parameter:

SI

Value:

3.2

Test group / Remarks:

1%

Parameter:

SI

Value:

8.1

Test group / Remarks:

2%

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

Details on the study design:

Test System
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Plating of Cells
For testing, cells were 80 - 90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage numbers used were 18, 20 and 22 in experiment 1, 2 and 3, respectively.

Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about
48 hours at 37 ±1.0 °C in the presence of 5% CO2. In total 3 experiments were performed.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37 °C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

Experiment 1:The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.58 and the EC1.5 33 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.45 and the EC1.5 39 µM.
Experiment 3:The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.37 and the EC1.5 46 µM.

Run / experiment:

other: Experiment 1

Parameter:

other: IC 50 (µg/ml )

Value:

38

Positive controls validity:

valid

Run / experiment:

other: Experiment 2

Parameter:

other: IC 50 µg/ml

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.

Run / experiment:

other: Experiment 3

Parameter:

other: IC50 µg/ml

Value:

86

Positive controls validity:

valid

Three independent experiments were performed. The cells were in these experiments incubated with the test item in concentration ranges of 0.20 - 400 µg/mL, 0.0005 – 1.0 µg/mL and 0.049 - 100 µg/mL for 48 hours in experiment 1, 2 and 3 respectively. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

Experiment 1

·          EnvaMul 600 showed toxicity. The calculated IC₃₀ was 34 µg/mL and the calculated IC₅₀ was 38 µg/mL. 

·          A dose related luminescence activity induction was observed after treatment with EnvaMul 600. The maximum luciferase activity induction (I_max) was 10.90-fold. Since a >1.5 fold induction was already observed at the lowest dose level tested of 0.20 µg/mL, no EC_1.5 could be calculated

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_max was 3.58 and the EC_1.5 33 µM. 

Experiment 2

·          EnvaMul 600 showed no toxicity in the range 0.0005 – 1.0 µg/mL. The viability of the cells was higher than 70% at all test concentrations and therefore, no IC₃₀ and IC₅₀ values could be calculated. 

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with EnvaMul 600. The I_max was 1.17 and therefore no EC_1.5 could be calculated. 

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_max was 2.45 and the EC_1.5 39 µM. 

Experiment 3

·          EnvaMul 600 showed toxicity. The calculated IC₃₀was 81 µg/mL and the calculated IC₅₀ was 86 µg/mL. 

·          A dose related luminesce activity induction was observed after treatment with the test item. The I_max was 3.42 and the EC_1.5 8.1 µg/mL.

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_max was 2.37 and the EC_1.5 46 µM. 

All three experiments passed the acceptance criteria:

·          The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. 

·          The EC_1.5 of the positive control was between 5 and 125 µM (33 µM, 39 µM and 46 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.58-fold, 2.45-fold and 2.37-fold in experiment 1, 2 and 3, respectively).

·          Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (7.4%, 6.2% and 5.1% in experiment 1, 2 and 3, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

Interpretation of results:

other: EnvaMul 600 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report

Conclusions:

EnvaMul 600 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of EnvaMul 600to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens Ô assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch HD0379UD11 of EnvaMul 600 was a viscous brown liquid. EnvaMul 600 was suspended in dimethyl sulfoxide at 40 mg/mL. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.20 – 400 µg/mL (2-fold dilution series). Due to induction of the luciferase activity, the test concentrations for the second experiment were 0.0005 – 1.0 µg/mL (2-fold dilution series). Since the first two experiments obtained different results a third experiment was performed. In the third experiment, a stock solution of 10 mg/mL was prepared. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted to test concentrations of 0.049 – 100 µg/mL (2-fold dilution series). The test item precipitated at the dose levels of 100, 200 and 400 µg/mL in the first experiment.  

All three experiments passed the acceptance criteria:

·          The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. 

·          The EC_1.5 of the positive control was between 5 and 125 µM (33 µM, 39 µM and 46 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.58-fold, 2.45-fold and 2.37-fold in experiment 1, 2 and 3, respectively).

·          Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (7.4%, 6.2% and 5.1% in experiment 1, 2 and 3, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

In the first experiment, the test item showed toxicity. The calculated IC₃₀ was 34 µg/mL and the calculated IC₅₀ was 38 µg/mL. A statistically significant induction of the luciferase activity (p<0.001 Student’s t test) was measured. The maximum luciferase activity induction (I_max) was 10.90-fold. Since a >1.5 fold induction was already observed at the lowest dose level tested of 0.20 µg/mL, the EC_1.5 was set at <0.20 µg/mL.

The test item was retested with a lower concentration range of 0.0005 – 1.0 µg/mL to determine the EC_1.5 value more precisely. In the second experiment, the test item showed no toxicity (no IC₃₀ and IC₅₀ value) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (I_max) was 1.17-fold. 

Since the first two experiments obtained different results a third experiment was performed. In the third experiment,the test item showed toxicity. The calculated IC₃₀ was 81 µg/mL and the calculated IC₅₀ was 86 µg/mL. In the third experiment, a statistically significant induction of the luciferase activity (EC_1.5 value 8.1 µg/mL; p<0.001 Student’s t test) was measured. The maximum luciferase activity induction (I_max) was 3.42-fold.

EnvaMul 600is classified as positive in the KeratinoSens^TM assay since positive results (>1.5-fold induction) were observed at test concentrations of <200 µg/mL with a cell viability of >70% compared to the vehicle control in two out of three experiments.

In conclusion, EnvaMul 600 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test substance was found being a skin sensitiser in an in vitro ARE-test as well as in an in vivo LLNA test. Based on in vivo test data, the substance is to be classified as a strong skin sensitiser, category 1A according to CLP (Regulation EC No 1272/2008).