Phenol, isopropylated, phosphate (3:1)

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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.

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

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.

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A Weight of Evidence approach is undertaken for Skin Sensitisation, based on a combination of study data, human health monitoring and QSAR results. The following is assessed:

 

Local Lymph Node Assay:

 

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.

Human Health Monitoring Data:

 

Medical data from the site of point manufacture has been reviewed for a period of 50 years, in order to assess sensitisation. In addition, research has determined a human patch test that assessed aryl phosphate exposure for both irritation and sensitisation. The data is considered appropriate for inclusion due to the fact that this is used as part of due diligence occupational exposure monitoring, and is an accurate representation of endpoint effects (lack of) at the point of key exposure. Both studies are presented together here as they are supportive of the occupational works conducted at the site.

 

On the basis of the available human data, both historical and study specific, it is concluded that the aryl phosphate substances manufactured by Chemtura do not cause skin sensitization in man. The evidence presented in the historical data on epidemiological effects detail that skin effects are not prevalent within the workforce involved in the production, handling and maintenance of the areas associated with the aryl phosphates. This conclusion is reinforced by the study specific data where irritation and sensitization effects in man where studied via the use of human patch testing. The results of this study where negative, with no effects seen in any of the volunteers assessed.

 

Aryl phosphates are therefore not considered to be skin sensitisers in man on the basis of the evidence available.

 

QSAR – DEREK Sensitisation Prediction

 

All isomers of IPTPP were analysed for skin sensitisation using Derek Nexus in a range of mammalian species. The results indicate that IPTPP does not contain any alerts for skin sensitisation. 

Using a later version of DEREK for Windows a review of a guinea pig archive data for compounds eliciting allergic contact dermatitis and a local lymph node assay (LLNA) data set was conducted. DEREK for windows correctly predicted 82.9% and 73% of the results from the guinea pig and LLNA data (Fedorowiczet al., 2005). New alerts are added each year to Derek software and this indicates increasing accuracy of the software with development of new versions.

 

The data provided by DEREK is considered to be substantial supporting evidence of the effects noted in the human exposure studies.

 

QSAR – CAESAR Sensitisation Prediction

 

 A QSAR assessment using the CAESAR project system for skin sensitisation was conducted. The CAESAR model for skin sensitization is based on a data set that includes 209 compounds extracted from the paper by Gerberick et al. As the system is not yet listed on the QSAR Model Reporting Format Inventory, it has been assigned a reliability of 2. This is based on the fact that the system has undergone extensive validation in the CAESAR validation process and is considered viable, but has not yet been accepted as an approved QSAR model. All possible 27 isomers of the substance have been evaluated using the system, and whereas some of the descriptors for this compound have values outside the descriptor range for the compounds of the training set, the model is considered to provide useful supporting information.

 All isomers of IPTPP were analysed for skin sensitisation using the CAESAR model for skin sensitization. The results indicate that IPTPP does not contain any alerts for skin sensitisation.

 

The data provided by CAESAR is considered to be substantial supporting evidence of the effects noted in the human exposure studies.

 

Overall conclusion:

 

The data provided is considered to be substantial supporting evidence of the effects noted in the human exposure studies. The substance is not considered to be a sensitiser on the basis of a weight of evidence approach.

 

 

 

Migrated from Short description of key information:

Skin sensitisation only is discussed below.  No direct studies on respiratory sensitisation are available; however experience in use would indicate that this hazard is not experienced with manufacture and use of the substance.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No data available. Experience in use would indicate that this hazard is not experienced with manufacture and use of the substance.

Migrated from Short description of key information:

No data available.

Justification for classification or non-classification

Justification for classification or non classification

The above studies have been ranked reliability 1 or 2 according to the Klimish et al system. Reliability 2 ranking was deemed appropriate to the main LLNA study was conducted to GLP in compliance with agreed protocols, but the results did not fulfil the definitive requirements of the OECD guideline. The DEREK QSAR prediction is considered reliability 1 as the methodology is well documented, and the results fell within the model system that was used in the assessment. The human health monitoring data was assigned reliability 1, based on the historical data that is available in support of the registration, plus the additional human patch testing that was conducted on the substance in the past. Finally, the CAESAR QSAR prediction is assigned as reliability 2, as there is no existing QMRF validated method available, although the CAESAR program has conducted extensive validation in conjunction with a number of parties.

Justification for classification or non classification

On the basis of the weight of evidence approach, the above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification or labelling is applicable for this endpoint.