1-[4-(1,1-dimethylethyl)phenyl]-3-(4-methoxyphenyl)propane-1,3-dione

Endpoint summary

Description of key information

The test article produced inconsistent results in various in vitro and in chemico sensitization assays, but was negative in a GMPT study with guinea pigs and in a GLP-compliant LLNA.

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:

key study

Study period:

08.12.2022-31.1.2023

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:

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:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc
- Microbiological status of animals, when known:
- Age at study initiation: 1st pre-test: 17 weeks; 2nd pre-test & main study: 8 - 12 weeks
- Weight at study initiation: 1st pre-test: 25 g (1 animal); 2nd pre-test: 17.1 g (1 animal) main study: 18.6 g average (16 animals)
- Housing: per group; Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet(certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 45-65%
- Air changes (per hr): 15
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:

dimethylformamide

Remarks:

The highest test item concentration, which could be technically used was a 50% solution in DMF. Vortexing was used to formulate the test item.

Concentration:

10%, 25%, 50%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in one animal. One mouse was treated by (epidermal) topical application to the dorsal surface of each ear with a test item concentration of 50% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled and weighed using an analytical balance. The animal showed an erythema of the ear skin (Score 1), and, after each application mild and unspecific signs of discomfort such as piloerection, partially closed eyes, decreased activity, and hunched posture. An erythema of ear skin could not always be determined due to substance residuals. Since a relatively high ear thickness value was determined on day 6, which was not accompanied by a similar increase in the other irritation parameters (i.e., ear weights, erythema score), a confirmatory second pre-test was performed in one further animal using a test item concentration of 50% again. The animal showed again mild and unspecific signs of discomfort such as decreased activity, hunched posture, nervousness, and partially closed eyes as well as slightly scaly ears. This time, however, all parameters for possible local skin irritation (ear weights, ear thickness, erythema score) were well within the Guideline-recommended thresholds. A possible erythema of ear skin could not always be determined due to test substance residuals.
Thus, the test item in the main study was assayed at 10, 25, and 50%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

- Ear Thickness: In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.
- Ear Weights: In the pre-test, after the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm²). For each animal both

MAIN STUDY
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10, 25, and 50% in DMF. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.4 µCi of 3H-methyl thymidine (equivalent to 81.4 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.

The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

- Clinical observation: All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.
punches were immediately weighed per animal using an analytical balance.
- Body weights: The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

Positive control substance(s):

other:

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Parameter:

SI

Value:

1

Test group / Remarks:

control

Parameter:

SI

Value:

1.17

Test group / Remarks:

10%

Parameter:

SI

Value:

1.48

Test group / Remarks:

25%

Parameter:

SI

Value:

1.8

Test group / Remarks:

50%

Cellular proliferation data / Observations:

EC3 CALCULATION
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were observed during the study period.
The animals treated with test item concentrations of 25 and 50% showed a very slight erythema of the ear skin (Score 1) on test day 3 only. Animals treated with 10% test item concentration did not show any signs of local skin irritation. An erythema of ear skin could not always be determined due to substance residuals.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	23	---	---	---	---
---	BG II	21	---	---	---	---
0	1	7722	7700	8	962.5	1.00
10	2	9042	9020	8	1127.5	1.17
25	3	11387	11365	8	1420.6	1.48
50	4	13903	13881	8	1735.1	1.80

1 = Control Group

2-4 = Test Group

a) = The mean value was taken from the figures BG I and BG II

b) =Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was not a skin sensitiser under the test conditions of this study.

Executive summary:

In this study the test item formulated in dimethylformamide (DMF) was assessed for its possible skin sensitising potential. For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. The animals treated with a test item concentration of 25 and 50% showed a very slight erythema of the ear skin (Score 1) on test day 3 only. Animals treated with 10% test item concentration did not show any signs of local skin irritation. In this study Stimulation Indices (S.I.) of 1.2, 1.5, and 1.8 were determined with the test item at concentrations of 10, 25, and 50% in DMF, respectively. The test item was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Various in vitro/in chemico assay results (included in the dossier as weight of evidence entries) are available for the test article showing inconsistent findings, as depicted in the table below:

Key Event 1		Key Event 2		Key Event 3
Test	Result	Test	Result	Test	Result
ADRA	negative	LuSens	positive	U-Sens	positive
DPRA	negative	KeratinoSens	negative	H-CLAT	positive
PPRA	positive

The test item was further tested negative in a SENS-IS assays. Overall, the existing in vitro data are not sufficient for a conclusion.

 

As a follow up, in line with ECHA's final decision requesting additional experimental data on the sensitizing potential of the test article, a Local Lymph Node Assay was performed. 

 

In this assay, three groups each of four female mice were treated once daily with the test item at concentrations of 10, 25, and 50% (w/w) in DMF by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments. A control group of four mice was treated with the vehicle (DMF) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a β-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. The animals treated with test item concentrations of 25 and 50% showed a very slight erythema of the ear skin (Score 1) on test day 3 only. Animals treated with 10% test item concentration did not show any signs of local skin irritation. In this study Stimulation Indices of 1.2, 1.5, and 1.8 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in DMF. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

 

The test item was not a skin sensitizer under the conditions of this study. This result is in line with the negative results reported in a GPMT study performed in 1982.

 

Overall, the test article is therefore not a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified as a skin sensitizer under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.