Hexaphenoxycyclotriphosphazene

Endpoint summary

Description of key information

Since HPCTP, tested in DMF, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay, no classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017.

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:

2019

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)

Version / remarks:

22 July 2010

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Batch/Lot number: 190202
Description: White or light yellow crystal
Purity: 99.53%
Expiry date: 31 January 2021
Storage conditions: Controlled room temperature (15-25 ºC, below 70% relative humidity).
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaCrl mice
Source: Charles River UK Limited, Manston Road, Margate Kent, CT9 4LT, United Kingdom
Hygienic level: SPF at arrival; standard housing conditions during the study
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 / group
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 8 weeks old (age-matched, within one week)
Body weight range at starting: 18.0 – 19.8 grams (The weight variation in animals in the study did notexceed ± 20% of the mean weight.)
Acclimatisation time: 13 days
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / Additional enrichment (hiding tunnels) wasalso used during the study.
Cage type: Type II. polypropylene / polycarbonate
Bedding and nesting: Bedding and nesting materials were available to animalsduring the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1 – 23.8°C
Relative humidity: 22 - 73%
Ventilation: 15-20 air exchanges/hour

Vehicle:

dimethylformamide

Concentration:

10%, 5%, 2.5%

No. of animals per dose:

4

Details on study design:

In the main assay, twenty female CBA/CaCrl mice were allocated to five groups, each group comprised four animals:
- three groups of animals received HPCTP (formulated in DMF) at 10, 5, and 2.5% (w/v), respectively,
- a negative control group received the vehicle (DMF) only,
- a positive control group received 25% (w/v) HCA (dissolved in DMF).

The test item solutions were applied to the dorsal surface of the ears of the experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3) and then maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minutes after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

Positive control substance(s):

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

Positive control results:

Positive controls responded as expected

Key result

Parameter:

SI

Value:

ca. 0.6

Test group / Remarks:

HPCTP 10%(w/v) in DMF

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Key result

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

HPCTP 5%(w/v) in DMF

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Key result

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

HPCTP 2.5%(w/v) in DMF

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

No test item residue was observed in any of the animals. There were no indications of any irritancy at the site of application. No marked body weight losses (≥5%) were observed during the study in any of the animals. The results of the proliferation assay are summarised in Table below. The appearance of the lymph nodes was normal in all animals.

Table: DPM, DPN and Stimulation IndexValues for all Groups

 

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5% (w/v)TCA)	33.5	-	-	-	-
Negative control (DMF)	4189	4155.5	8	519.4	1.0
HPCTP 10%(w/v) in DMF	2717	2683.5	8	335.4	0.6
HPCTP 5%(w/v) in DMF	4045	4011.5	8	501.4	1.0
HPCTP 2.5%(w/v) in DMF	4226	4192.5	8	524.1	1.0
Positive control(25%(w/v) HCA in DMF)	24456	24422.5	8	3052.8	5.9

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of the present assay, HPCTP, tested in DMF, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay. No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of HPCTP following dermal exposure in mice. The study was performed with vertebrate animals as classification by use of in vitro alternatives was not possible for this test item. The minimum number of animals was used, corresponding to the regulatory guidelines being followed. The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD 429 vehicles were assessed: Acetone: Olive oil 4:1 (v/v) mixture, N,N-dimethylformamide (abbreviated as DMF), Methyl ethyl ketone, Propylene glycol, Dimethyl sulfoxide, 1% aqueous Pluronic® PE9200, and cyclohexane. The best vehicle taking into account the test item characteristics, and the requirements of the relevant OECD guideline was considered to be DMF. The 10% (w/v, i.e. 0.1 g per mL with added vehicle) format was the highest concentration which was suitable for the preliminary test. The 10% and 5% (w/v) formulations appeared to be solutions by visual examination. The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaCrl mice using two doses (2 animals/dose) with the concentrations of 10% (w/v) and 5% (w/v) in DMF. Based on the results, 10% (w/v) was selected as top dose for the main test.

In the main assay, twenty female CBA/CaCrl mice were allocated to five groups, each group comprised four animals: - three groups of animals received HPCTP (formulated in DMF) at 10, 5, and 2.5% (w/v), respectively, - a negative control group received the vehicle (DMF) only, - a positive control group received 25% (w/v) HCA (dissolved in DMF). The test item solutions were applied to the dorsal surface of the ears of the experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3) and then maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minutes after the incorporation of tritiated methyl thymidine ( 3 HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group. There was no mortality or signs of systemic toxicity observed during the study. No marked body weight losses (≥5%) were observed during the study in any of the animals. The stimulation index values were 0.6, 1.0 and 1.0 at concentrations of 10, 5 and 2.5% (w/v), respectively. The size of lymph nodes was in good correlation with this conclusion. The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was 5.9, therefore demonstrating the appropriate performance of the assay. In conclusion, under the conditions of the present assay, HPCTP, tested in DMF, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay. No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted on 04 February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Batch No.: 190202
Description: white powder
Storage conditions: At room temperature, Protected from light
Molecular weight: 693.56 g/mol
Specific requirements (handling conditions): None
Purity: . 99.53%
Correction factor: No correction factor

Details on the study design:

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours, protected from light. At the end of the incubation, the concentrations of residual peptides were evaluated by liquid chromatography with Ultra-Violet (HPLC/UV) detection at 220 nm. Peptide reactivity was reported as percentage depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate vehicle).

Positive control results:

Positive controls responded as expected

Key result

Parameter:

other: mean depletion value

Remarks:

for cysteine peptide

Value:

3.66

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Parameter:

other: mean depletion

Remarks:

for lysine peptide

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item HPCTP, was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation with the peptides.

Executive summary:

The objective of this study was to evaluate the reactivity of the test item, HPCTP, to synthetic cysteine and lysine peptides. This test addresses the molecular initiating event of the skin sensitisation adverse outcome pathway (AOP) and is part of a tiered strategy for skin sensitization assessment. The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours, protected from light. At the end of the incubation, the concentrations of residual peptides were evaluated by liquid chromatography with Ultra-Violet (HPLC/UV) detection at 220 nm. Peptide reactivity was reported as percentage depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate vehicle). The test item was dissolved at 100 mM in a 1:1 (v/v) mixture of acetone:acetonitrile. All acceptance criteria were met, the study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with both peptides. As a result, the mean percentage depletion values were calculated as follows: . for the cysteine peptide, the mean depletion value was 3.66%, . for the lysine peptide, the mean depletion value was set to 0% due to negative depletion. The mean of the percentage cysteine and percentage lysine depletions was equal to 1.83%. Accordingly, the test item was considered have no or minimal. Therefore, the DPRA prediction is considered as negative. Since precipitate was observed at the end of the incubation with the peptides, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity cannot be drawn with sufficient confidence.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019

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)

Version / remarks:

June, 2018

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Batch (Lot) Number: 190202
Expiry date: 31 January 2021 (expiry date)
Physical Description: White powder
Purity/Composition: See Certificate of Analysis
Storage Conditions: At room temperature

Details on the study design:

1. 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 number used was P+12 in experiment 1 and P+14 in experiment 2.
2. 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 covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0oC in the presence of 5% CO2. In total 2 experiments were performed.
3. Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the SteadyGlo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 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).
4. 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; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

Positive controls responded as expected

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: I max

Remarks:

Luminescence Induction

Value:

1.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: I max

Remarks:

Luminescence Induction

Value:

1.34

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Experiment 1:

- Precipitation was observed at the start and end of the incubation period in the 96-well plates at dose levels of 63 µM and upwards.

- HPCTP showed no toxicity. 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 HPCTP. The Imax was 1.14 and therefore no EC1.5 could be calculated.

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.23 and the EC1.5 33 µM.

Experiment 2:

- Precipitation was observed at the start and end of the incubation period in the 96-well plates at dose levels of 63 µM and upwards. -

- HPCTP showed no toxicity. 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 HPCTP. The Imax was 1.34 and therefore no EC1.5 could be calculated.

-The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.01 and the EC1.5 32 µM.

Interpretation of results:

other:

Conclusions:

HPCTP is classified as inconclusive under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of HPCTP to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The study procedures described in this report were based on the most recent OECD guideline. Batch 190202 of HPCTP was a white powder. The test item was dissolved in dimethyl sulfoxide at 25 mM. 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 0.12 – 250 µM (2-fold dilution series). The highest test concentration was considered to be the limit of solubility. The test item precipitated at dose levels of 63 µM and upwards. Two independent experiments were performed. Both experiments passed the acceptance criteria: - The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. - The EC1.5 of the positive control was within two standard deviations of the historical mean (33 µM and 32 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (4.23-fold and 3.01-fold in experiment 1 and 2, respectively). - Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.7% and 6.3% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.14-fold and 1.34-fold in experiment 1 and 2 respectively. HPCTP is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5 -fold induction) were observed at test concentrations < 1000 uM.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The weight of evidence approach was performed based on in chemico and in vitro data, addressing each of the key events of skin sensitization on its own or together. The information obtained in the in vitro skin sensitization test strategy is too limited for a reliable conclusion on the skin sensitization properties of HPCTP. Therefore, it is, as a last resort, justified to continue with an in vivo tests to determine and classify the skin sensitization properties of HPCTP.

Justification for classification or non-classification

HPCTP did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.