Reaction Mass of Distillates (petroleum), hydrotreated heavy paraffinic and Residual oils (petroleum), solvent refined; oxidized, calcium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 December 2016 to 24 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

Confidential

IUPAC Name:

Confidential

Test material form:

liquid: viscous

Details on test material:

- Appearance/physical state: Dark brown extremely viscous liquid
- Storage conditions: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Study design: in vivo (LLNA)

Vehicle:

other: butanone

Concentration:

25 %, 10 % or 5 % test item in butanone

No. of animals per dose:

Five

Details on study design:

TEST ITEM PREPARATION AND ANALYSIS
- The test item was freshly prepared as a solution in butanone. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The vehicle determination record
is included as Annex 3 (attached).
- The test item was formulated within 2 hours of being applied to the test system. It is assumed
that the formulation was stable for this duration.
- No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

REFERENCE ITEM PREPARATION
- The positive control item was freshly prepared as a 15 % v/v dilution in butanone.

ANIMAL INFORMATION
- Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands.
- On receipt, the animals were randomly allocated to cages.
- The animals were nulliparous and non-pregnant.
- After an acclimatisation period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card.
- At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

ANIMAL CARE AND HUSBANDRY
- Animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
- Temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively.
- The rate of air exchange was at least fifteen changes per hour.
- Lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
- The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

PRELIMINARY SCREENING TEST
- Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at a maximum attainable concentration of 25 % w/w in butanone, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Annex 4 (attached). Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
- The thickness of each ear was measured using a Mitutoyo 547-3008 gauge (Mitutoyo Corporation), pre and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN TEST
- Groups of five mice were treated with the test item at concentrations of 25 %, 10 % or 5 % w/w in butanone. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
- A further group of five mice received the vehicle alone in the same manner. The positive control animals were similarly treated to the test animals except that 25 μL of the positive control item, a-Hexylcinnamaldehyde, tech., 85%, at a concentration of 15 % v/v in butanone, was applied to the dorsal surface of each ear.
- Local skin irritation was scored daily according to the scale included as Annex 4. The thickness of each ear was measured and recorded pre and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6.

3H-METHYL THYMIDINE ADMINISTRATION
- Five days following the first topical application of the test item, vehicle control item or positive control item (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.

OBSERVATIONS
- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

TERMINAL PROCEDURES
- Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
- Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).
- Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by P-scintillation counting. The "Poly Q" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number ofradioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

DATA EVALUATION
- The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
- The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

MAJOR COMPUTERISED SYSTEMS
- Delta Controls - ORCA view
- R version 3.0.2 (2013-09-25) - "Frisbee Sailing"

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

STATISTICAL ANALYSIS
- Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett's multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.
- Probability values (p) were presented as *** (p < 0.001); ** (p < 0.0); * (p < 0.05); not significant (p ≥ 0.05).

Results and discussion

Positive control results:

RESULTS WITH POSITIVE CONTROL ITEM
- The Stimulation Index expressed as the mean radioactive incorporation for the positive control was reported as 5.02 (15 % v/v positive control item in butanone; positive).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.71

Test group / Remarks:

5 % w/w in butanone

Remarks on result:

other: negative

Results 2

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

10 % test item in butanone

Remarks on result:

other: negative

Results 3

Key result

Parameter:

SI

Test group / Remarks:

25 % test item in butanone

Remarks on result:

other: negative

Cellular proliferation data / Observations:

PRELIMINARY SCREENING TEST
- Clinical observations, body weight and mortality data are given in Appendix 1 (attached).
- Local skin irritation is given in Appendix 2 (attached).
- The ear thickness measurements and mean ear thickness changes are given in Appendix 3 (attached).
- No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25 % increase in mean ear thickness were noted.
- Based on this information the dose levels selected for the main test were 25 %, 10 % and 5 % w/w in butanone.

ESTIMATION OF PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS IN MAIN TEST
- The radioactive disintegrations per minute per lymph node and the stimulation index are given in Appendix 4 (attached).
- The Stimulation Index expressed as the mean radioactive incorporation for each treatment
group divided by the mean radioactive incorporation of the vehicle control group was reported as 1.71 (5 % test item w/w butanone; negative), 1.40 (10 % test item w/w butanone; negative), 2.14 (25 % test item w/w butanone; negative).

CLINICAL OBSERVATIONS AND MORTALITY DATA
- Individual clinical observations and mortality data for test and control animals are given in Appendix 5 (attached).
- Local skin irritation is shown in Appendix 6 (attached).
- The ear thickness measurements and mean ear thickness changes are given in Appendix 7 (attached).
- There were no unplanned animal deaths during the study.
- No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHT
- Individual body weights and body weight change for test and control animals are given in Appendix 8 (attached).
- Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a non-sensitiser under the conditions of the study. The positive control a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 15% v/v in butanone, thus, demonstrating the sensitivity and reliability of the test system.

Executive summary:

GUIDELINE

An investigation was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was conducted in compliance with OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) and Method B.42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

 

METHODS

Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable concentration of 25 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in butanone at concentrations of 25%, 10% or 5% w/w. A further group of five animals was treated with butanone alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, a-Hexylcinnamaldehyde tech., 85%, at a concentration of 15 % v/v in butanone.

 

RESULTS

The Stimulation Index expressed as the mean radioactive incorporation for each treatment

group divided by the mean radioactive incorporation of the vehicle control group was reported as 1.71 (5 % test item w/w butanone; negative), 1.40 (10 % test item w/w butanone; negative), 2.14 (25 % test item w/w butanone; negative). The Stimulation Index expressed as the mean radioactive incorporation for the positive control was reported as 5.02 (15 % v/v positive control item in butanone; positive).

 

CONCLUSION

The test item was considered to be a non-sensitiser under the conditions of the study. The positive control a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 15% v/v in butanone, thus, demonstrating the sensitivity and reliability of the test system.