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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:
weight of evidence
Study period:
04 August 2010 - 14 September 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant, carried out according to recognised guideline. However, a dose-response relationship was not observed, which in accordance with OECD guideline 429 is required of the LLNA test design. Hence a rating of 2 is applied on this basis.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Reofos 65 (Phenol, isopropylated, phosphate)
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: not specified
- Physical state: not specified
- Analytical purity: not specified
- Impurities (identity and concentrations): not specified
- Composition of test material, percentage of components: not specified
- Isomers composition: not specified
- Purity test date: not specified
- Lot/batch No.: 2009176176
- Expiration date of the lot/batch: 23 April 2011
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Stability under test conditions: not specified
- Storage condition of test material: roome temperature
- Other:

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J Rj
Sex:
female
Details on test animals and environmental conditions:
EXPERIMENTAL ANIMALS

Species and strain: CBA/J Rj mice
Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions


Justification of strain:On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / treatment group
Sex: female, nulliparous, non pregnant
Age of animals at starting: 8 – 9 weeks old
Body weight range at starting: 20.0 – 21.6 grams (The weight variation in animals involved in the study did not exceed +/- 20 % of the mean weight)
Acclimatization time:6 days


Husbandry

Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Individual caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene/ polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 15-20 air exchange/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.

Room/Cabinet (non-radioactive phase): 244/4
Room/Cabinet (radioactive phase): 139 - 140


Food and feeding

Animals received ssniff SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 847 4257 Expiry Date: 11 January 2011) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum. The contents of the standard diet are detailed in Appendix 2.


Water supply

Animals received tap water from the municipal supply from 500 ml bottle, ad libitum.
Water quality control analysis was performed once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at LAB Research Ltd.


Bedding

Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberger (Germany) Holzmühle 1) was available to animals during the study.


Identification

A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of LAB Research Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software according to the actual body weights, verifying the homogeneity and variability between the groups.


Study design: in vivo (non-LLNA)

Induction
Concentration / amount:
Not applicable.
Challenge
Concentration / amount:
Not applicable.
No. of animals per dose:
Not applicable.
Details on study design:
Not applicable.
Challenge controls:
Not applicable.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100 % w/v
No. of animals per dose:
4
Details on study design:
ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
A Preliminary Irritation/Toxicity Test was performed on CBA/J Rj mice using two doses, at test item concentrations of 100 (undiluted) and 50 (w/v) %, respectively. This preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 with a body weight measurement and radioactive proliferation assay was not performed.
During the Preliminary Irritation / Toxicity Test no mortality, systemic toxicity or local toxicity were observed.

Topical application
During the assay each mouse was topically dosed on the dorsal surface of each ear with 25 ul of the appropriate formulation applied using a pipette. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

PROLIFERATION ASSAY

Injection of Tritiated Thymidine (3H-TdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 l of sterile PBS (phosphate buffered saline) containing approximately 20 Ci of 3H-TdR using a gauge 25G1" hypodermic needle with 1 ml sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (+/- 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 ml) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) wase prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 ml). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 ml of PBS was added to the tubes. The washing step was repeated twice.
This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final wash, supernatant were removed leaving a small volume (<0.5 ml) of supernatant above each pellet. Each pellet was gently agitated before suspending the LNCs in 3 ml of 5% TCA (trichloroacetic acid) for precipitation of macromolecules. After incubation with 5% TCA at 2-8 deg C overnight (approximately 18 hours) precipitate was recovered by centrifugation at 190 x g for 10 minutes, supernatants were removed and pellets were suspended in 1 ml of 5% TCA and dispersed using ultrasonic water bath. Each precipitate was transferred to a suitable sized scintillation vial with 10 ml of scintillation liquid and thoroughly mixed. The vials were loaded to a β-scintillation counter and 3H-TdR incorporation was measured for up to 10 minutes per sample.
The β-counter expresses the 3H-TdR incorporation as the number of radioactive disintegrations per minute (DPM). Similarly, background 3H-TdR levels were also measured in two 1 ml aliquots of 5% TCA.

OBSERVATIONS

Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3H-TdR injection) with a precision of ± 0.1 g.

EVALUATION OF THE RESULTS
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 (w/v) % TCA solutions was used as the background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated.
A stimulation index of 3 or greater is an indication of a positive result.

Interpretation of Results
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3H-TdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

USE OF RADIOACTIVE MATERIALS
Use of radioactive materials was recorded in the appropriate register. Regular decontamination of the working area with a verification of decontamination was carried out. Radioactive waste materials were processed according to normal laboratory standards.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None

Results and discussion

Positive control results:
A significant lymphoproliferative response (stimulation index value of 29.2) was noted for a-Hexylcinnamaldehyde in this experiment. The results of the positive control group demonstrated the appropriate performance of the assay.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
> 7.4 - < 12.9
Test group / Remarks:
25, 50 & 100%
Parameter:
other: disintegrations per minute (DPM)
Value:
> 2 167.5 - < 3 750.5
Test group / Remarks:
25, 50 & 100%
Parameter:
EC3
Value:
5.3
Test group / Remarks:
25 & 50%
Cellular proliferation data / Observations:
Clinical observation
No mortality or signs of systemic toxicity were observed during the study.

Body weight measurement
No treatment related effects were observed on animal body weights.

Any other information on results incl. tables

Table 3:Individual Body Weights for all Animals with Group Means

Animal

Number

Identity

Number

Test Group

Name

Initial Body

Weight (g)

Terminal Body

Weight (g)

5693

1

Negative control (vehicle):

21.2

21.1

5696

2

AOO

21.4

20.5

5686

3

 

20.5

20.6

5682

4

 

20.9

21.6

 

 

Mean

21.0

21.0

5701

5

Reofos 65

21.3

21.8

5697

6

100 % (undiluted)

21.6

23.1

5690

7

 

20.0

22.3

5705

8

 

20.6

22.6

 

 

Mean

20.9

22.5

5703

9

Reofos 65

21.1

21.9

5688

10

50 (w/v) % in AOO

21.4

21.5

5684

11

 

20.2

20.5

5695

12

 

20.6

21.1

 

 

Mean

20.8

21.3

5708

13

Reofos 65

21.6

24.1

5691

14

25 (w/v) % in AOO

21.3

21.6

5685

15

 

20.5

20.1

5683

16

 

20.2

20.7

 

 

Mean

20.9

21.6

5699

17

Positive control

21.6

21.6

5706

18

25 % HCA in AOO

21.6

22.5

5694

19

 

20.6

20.9

5700

20

 

20.6

22.0

 

 

Mean

21.1

21.8

 

Table 4:DPM, DPN and Stimulation Index Values for all Groups

Test Group

Measured

 

No. of

 

Stimulation

Name

DPM/group

DPM

Node

DPN

Index Values

Background

 

(5 (w/v) % TCA )

37.5

 

-

 

 

Negative control

 

AOO

329

291.5

8

36.4

1.0

Reofos 65

 

100 % (undiluted)

3078

3040.5

8

380.1

10.4

Reofos 65

 

50 % in AOO

3788

3750.5

8

468.8

12.9

Reofos 65

 

25 % in AOO

2205

2167.5

8

270.9

7.4

Positive control

 

25 % HCA in AOO

8547

8509.5

8

1063.7

29.2

Applicant's summary and conclusion

Interpretation of results:
ambiguous
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the present assay Reofos 65 (Batch No.: 2009176176), tested in a suitable vehicle, was shown to be ambiguous for sensitization potential in the Local Lymph Node Assay, as no dose-response relationship was observed within the study.
Executive summary:

Under the conditions of the study, Stimulation Indices (SI) of 7.4, 12.9 and 10.4 were calculated for applied concentrations of 25%, 50% and 100% IPTPP, respectively.  Considering that the SI threshold value, as stated in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC, is at least 3, the substance would normally be classified as a sensitizer.  However, a dose-response relationship was not observed, which in accordance with OECD guideline 429 is required of the LLNA test design.  That is, the guideline states,’ [t]he decision process with regard to a positive response includes a stimulation index ≥3, together with consideration of dose-response and, where appropriate, statistical significance.’

Given that supporting data on sensitization all indicates that aryl phosphates do not cause sensitization, and that a dose-response relationship was no observed in this test, it is considered that the results of the study are inconclusive.