2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropanoyl)phenoxy]phenyl]-2-methylpropan-1-one

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2019-01-21 - 2019-03-14 (experimental phase)

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: test system / method was potentrially unsuitable for the test item and the effect of no clear dose-response curve in all experiments could be an indication of a false positive result

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted: 25. June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU-Method B.60 of the Commission Regulation (EU) No. 2017/735: “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method

Version / remarks:

Adopted 14. Feb. 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD. Performance standards for assessment of proposed similar or modified in vitro skin sensitisation ARE-Nrf2 Luciferase test methods. ENV/JM/MONO(2015)6

Version / remarks:

22. May 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

This in vitro study was performed to assess the potential of the test item 2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one to activate the Nrf2 transcription factor by using the genetically modified keratinocyte cell-line “LuSens” (Bauch et al. 2012).
It employs the use of a reporter gene for luciferase placed under the control of the antioxi-dant response element (ARE) and hence monitors Nrf2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signalling pathway (Ade et al. 2009, Natsch 2012, Natsch & Emter 2008, Vandebriel et al. 2010). In order to conclude on the Nrf2 transcription factor activity of the test substance, at least two, but a maximum of three independent and valid experiments are performed.
The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible.

Positive control results:

The positive control induced a clear effect with an induction value of 4.9 fold in experiment 1, 5.9 fold in experiment 2, 5.8 fold in experiment 3 and 6.2 fold in experiment 4, in comparison to the solvent control.

Run / experiment:

other: Experiment I, II and V; test item concentrations: 26.9 µM, 32.3 µM, 38.8 µM, 46.5 µM, 55.8 µM, 67.0 µM, 80.4 µM, 96.5 µM, 115.7 µM

Parameter:

other: Relative Viability %

Value:

71.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment IV; test item concentrations 26.9 µM, 32.3 µM, 38.8 µM, 46.5 µM, 55.8 µM, 67.0 µM, 80.4 µM, 96.5 µM, 115.7 µM, 138.9 µM

Parameter:

other: Relative Viability %

Value:

72.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Interpretation of results:

study cannot be used for classification

Conclusions:

The study was performed according to OECD TG 442D under GLP on the registered substance itself. Positive and negative controls were valid.
The ARE-Nrf2 Luciferase Test addresses the second key event of the skin sensitisation adverse outcome pathway (AOP) that was defined by the OECD in 2012 and is a part of the AOP-based “two out of three” skin sensitisation integrated testing strategy for hazard identification (Bauch et al., 2012).
Under the experimental conditions of this study, the test item 2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor. However, the effect of no clear dose-response curve in all experiments could be an indication of a false positive result.

Executive summary:

A study was performed to assess the potential of the test substance to cause skin sensitisation in an ARE-Nrf2 luciferase test using the LuSens cell line according to OECD Guideline 442D, in compliance with GLP. The assay was performed as four independent experiments. As no dose-response was observed in the two first experiments (I and II), they were repeated to verify whether this was specific to the test substance or due to an artefact. Twelve concentrations were used in all experiments (26.9, 32.3, 38.8, 46.5, 55.8, 67.0, 80.4, 96.5, 115.7, 138.9, 166.7 and 200.0 µM), with an exposure time of 48 h. None of the actual concentrations (adjusted to the amount weighed) deviated by more than 1.3% from nominal values and test substance precipitation was not visible at any dose level. EGDMA (120 µM) was used as positive control, DL-lactic acid (5000 µM) as negative control and there was a luciferase induction growth control. Since all acceptability criteria of the assay were met, the study was considered valid. In experiments I, II and V, cytotoxic effects were observed at 138.9, 166.7 and 200.0 µM. In experiment IV, the two highest concentrations (166.7 and 200.0 µM) showed cytotoxicity. These cytotoxic concentrations were excluded from the evaluation of luciferase induction. Finally, the following test substance concentrations showed a viability ≥70% and could be evaluated for luciferase induction:  

 

-         Experiment I, II and V: 26.9, 32.3, 38.8, 46.5, 55.8, 67.0, 80.4, 96.5 and 115.7 µM;    

-         Experiment IV: 26.9, 32.3, 38.8, 46.5, 55.8, 67.0, 80.4, 96.5, 115.7 and 138.9 µM.  

 

 A statistically significant ≥1.5 -fold increase in luciferase induction in comparison to the solvent control was measured at all non-cytotoxic test substance concentrations. In experiments I and II, no clear dose-response was observed. The same was true for the follow-up experiments IV and V. In conclusion, under the experimental conditions of the LuSens assay, the test substance was positive. However, the lack of a clear dose-response curve in all experiments could be an indication of a false positive result (Holler, 2019). The study is therefore considered to be inconclusive. 

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July - 12 August 2005 (experimental period)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

background for conducting guinea pig maximisation test: unknown

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- 10/5 (test/control) Hsd Poc: DH - guinea pigs (Full-Barrier), Sex: female, nulliparous, nonpregnant, bodyweight at the commencement of the study 300 - 500 g.
- 3 (range finding) Hsd Poc: DH - guinea pigs (Full-Barrier), Sex: female, nulliparous, nonpregnant, bodyweight at the commencement of the study 300 - 500 g.
- A health inspection was performed to ensure the good state of health of the animals.
- The animals were derived from a controlled full barrier maintained breeding system (SPF).
- Source: Harlan Winkelmann GmbH, Borchen, Germany.

ENVIRONMENTAL CONDITIONS
- The animals were barrier maintained (semi-barrier) in an air conditioned room.
- Temperature: 22 +/- 3 °C
- Rel. humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Feeding ad libitum, Altromin 3122 maintenance diet for guinea pigs, rich in crude fiber, totally-pathogen-free (TPF)
- Free access to tap water (drinking water, municipal residue control, microbiol. controlled periodically)
- The animals were kept in groups in Terluran-cages on Altromin saw fiber bedding.
- Adequate acclimatization period.

Route:

intradermal and epicutaneous

Vehicle:

cotton seed oil

Concentration / amount:

- intradermal injection (induction - first stage): test item was applied at a 5% concentration (diluted in cotton seed oil).
- topical application (induction - second stage): test item was applied at a 50% concentration (diluted in cotton seed oil).

Day(s)/duration:

day 0: intradermal treatment; day 6: pretreatment of the test area with 0.5 ml of 10% sodium lauryl sulphate; day 7: occlusive dressing for 48 hours (test group: 0.5 mL of the prepared test item; control group: 0.5 mL of the vehicle)

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Route:

epicutaneous, occlusive

Vehicle:

cotton seed oil

Concentration / amount:

topical application (challenge): test item was applied at a 50% concentration (diluted in cotton seed oil).

Day(s)/duration:

day 20 (test and control group): 0.5 mL of the prepared test item was applied to the left flank of the animals and, a patch loaded with 0.5 mL vehicle to the right flank (intraspecific control); occlusive dressing for 24 hours.

No. of animals per dose:

- test group: 10 animals
- control group: 5 animals
- range finding: 3

Details on study design:

RANGE FINDING TEST:
For the justification of dose levels a preliminary test was performed. One animal was intradermally treated with 2.5 as well as 5% concentration (diluted in cotton seed oil, Lot 015K0202, Sigma) of the test item.
For the 2.5% concentration erythema grade 1 was observed (24 and 48 hours post-dose). After 72 hours no signs of irritation were observed any more.
For the 5% concentration erythema grade 1 was observed (24, 48 as well as 72 hours post-dose).
Therefore the concentration of 5% was chosen for the intradermal induction.
Two animals were topically treated with 50 as well as 100% concentration for 24 hours and 48 hours, respectively.
No signs of irritation and systemic toxicity were recorded for any concentration 24, 48 as well as 72 hours after application.
Therefore the 50% concentration was chosen for the topical induction as well as for the challenge.

MAIN STUDY
A. INDUCTION (First Stage, Intradermal Injection)

Three pairs of intradermal injections of 0.1 mL volume were given in the shoulder region which was cleared of hair by clipping so that one of each pair lies on each side of the midline.

Test group: Day 0
- Injection 1: Freund's Adjuvant complete, 1 + 1 (v/v) diluted with isotonic saline
- Injection 2: Prepared test item
- Injection 3: Prepared test item at a concentration of 50% (V/V) in Freund's Adjuvant complete, 1 + 1 (v/v) diluted with isotonic saline

Control group : Day 0
- Injection 1: Freund's Adjuvant complete, 1 + 1 (v/v) diluted with isotonic saline
- Injection 2: Vehicle at a concentration of 100%
- Injection 3: Vehicle at a concentration of 50% (V/V) in Freund's Adjuvant complete, 1 + 1 (v/v) diluted with isotonic saline
- Injections 1 and 2 were given close to each other and nearest to the head, while 3 is given toward the caudal part of the test area.

B. INDUCTION (Second Stage, Topical Application):

Test and Control Group: Day 6
Approximately twenty-four hours before the topical induction application the test area was closely clipped and painted with 0.5 mL of 10% sodium lauryl sulfat.

Test Group: Day 7
A patch was fully loaded with 0.5 mL of the prepared test item, applied to the test area and held in contact by an occlusive dressing for 48 hours.

Control Group: Day 7
A patch was fully loaded with 0.5 mL of the vehicle and applied to the test area and held in contact by an occlusive dressing for 48 hours.

C. CHALLENGE (Topical Application)

The flanks of treated and control animals were cleared of hair by closely-clipping.

Test and Control Group: Day 20
A patch loaded with 0.5 mL of the prepared test item was applied to the left flank of the animals and, a patch loaded with 0.5 mL vehicle to the right flank (intraspecific control), respectively. The patches were held in contact by an occlusive dressing for 24 hours.

Challenge controls:

A patch loaded with 0.5 mL of the prepared test item (50% in cotton seed oil) was applied to the left flank of the animals and, a patch loaded with 0.5 mL vehicle to the right flank (intraspecific control), respectively. The patches were held in contact by an occlusive dressing for 24 hours.

Positive control substance(s):

yes

Remarks:

reliability of the test system was performed in Jan./Feb. 2005 with Mercaptobenzothiazole [purity > 98%; CAS No. 149-30-4; Lot S35857 306; Merck] (15% in Vaseline)

Positive control results:

Frequency of sensitisation in positive control animals:
- at 24 h 80%,
- at 48 h 80%,
- at 72 h 60%.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no visible clinical symptoms over the period of observation

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no visible clinical symptoms over the period of observation

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

15%

No. with + reactions:

8

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

15%

No. with + reactions:

8

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

other: EU-GHS criteria not met

Conclusions:

The study was performed as GLP guideline study with no deviations and is well-documented. Hence, the results are considered reliable for assessment. The test item caused no reactions identified as sensitization.

Executive summary:

A study was performed to assess the potential of the test substance to cause skin sensitisation in guinea pigs according to OECD Guideline 406, in compliance with GLP. Groups of 10 Hsd Poc: DH guinea pigs (Full-Barrier) were used for the study, along with groups of 5 controls. The maximum concentration not giving rise to irritating effects in the preliminary test corresponded to 100% test substance. The first induction was conducted with 5% test substance in cottonseed oil, the second with 50% test substance in cottonseed oil. The concentration of test substance and vehicle used at each stage of challenge was equivalent to 50%. The study results were as follows:

• Induction I: 24 h post-dose erythema grade 1 at the injection site 2 in 8 animals; 48 h post-dose erythema grade 1 at the injection site 2 in 8 animals;
• Induction II: No signs of irritations in any animal.
• Challenge: No evidence of sensitization. No other effects observed.

 Under the study conditions, the test substance was considered to be non-sensitising (Brummer, 2005).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In vitro  

 

A study was performed to assess the potential of the test substance to cause skin sensitisation in an ARE-Nrf2 luciferase test using the LuSens cell line according to OECD Guideline 442D, in compliance with GLP. The assay was performed as four independent experiments. As no dose-response was observed in the two first experiments (I and II), they were repeated to verify whether this was specific to the test substance or due to an artefact. Twelve concentrations were used in all experiments (26.9, 32.3, 38.8, 46.5, 55.8, 67.0, 80.4, 96.5, 115.7, 138.9, 166.7, and 200.0 µM), with an exposure time of 48 h. None of the actual concentrations (adjusted to the amount weighed) deviated by more than 1.3% from nominal values and test substance precipitation was not visible at any dose level. EGDMA (120 µM) was used as positive control, DL-lactic acid (5000 µM) as negative control and there was a luciferase induction growth control. Since all acceptability criteria of the assay were met, the study was considered valid. In experiment I, II and V, cytotoxic effects were observed at 138.9, 166.7 and 200.0 µM. In experiment IV, the two highest concentrations (166.7 and 200.0 µM) showed cytotoxicity. These cytotoxic concentrations were excluded from the evaluation of luciferase induction. Finally, the following test substance concentrations showed a viability ≥70% and could be evaluated for luciferase induction:  

 

• Experiment I, II and V: 26.9, 32.3, 38.8, 46.5, 55.8, 67.0, 80.4, 96.5, and 115.7 µM;  
• Experiment IV: 26.9, 32.3, 38.8, 46.5, 55.8, 67.0, 80.4, 96.5, 115.7, and 138.9 µM.  

 

A statistically significant increase ≥1.5-fold in luciferase induction in comparison to the solvent control was measured at all non-cytotoxic test substance concentrations. In experiments I and II, no clear dose-response was observed. The same was true for the follow-up experiments IV and V. In conclusion, under the experimental conditions of the LuSens assay, the test substance was positive. However, the lack of a clear dose-response curve in all experiments could be an indication of a false positive result (Holler, 2019). The study is therefore considered to be inconclusive. 

     

In vivo  

 

A study was performed to assess the potential of the test substance to cause skin sensitisation in guinea pigs according to OECD Guideline 406, in compliance with GLP. Groups of 10 Hsd Poc: DH guinea pigs (Full-Barrier) were used for the study, along with groups of 5 controls. The maximum concentration not giving rise to irritating effects in the preliminary test corresponded to 100% test substance. The first induction was conducted with 5% test substance in cottonseed oil, the second with 50% in test substance in cottonseed oil. The concentrations of test material and vehicle used at each stage of challenge was equivalent to 50%. The study results were as follows:

 

• Induction I: 24 h post-dose erythema grade 1 at the injection site 2 in 8 animals; 48 h post-dose erythema grade 1 at the injection site 2 in 8 animals;
• Induction II: No signs of irritations in any animal.
• Challenge: No evidence of sensitization. No other effects observed.

 

Under the study conditions, the test substance was therefore considered to be non-sensitising (Brummer, 2005).  

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of a guinea pig maximisation test (OECD Guideline 406), the test substance does not meet the CLP classification criteria with respect to skin sensitization, as set out in Regulation (EC) No. 1272/2008.