4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Endpoint summary

Description of key information

- Skin sensitisation: sensitiser, females, mice, Local Lymph Node Assay, OECD 429, Pooles 2017

- Respiratory sensitisation: not expected to be of concern for respiratory sensitisation

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:

26 Feb 2016 to 16 Mar 2016

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:

July 2010

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Nulliparous and non-pregnant
- Age at study initiation: 8 - 12 weeks
- Body weight at study initiation: 15 - 23 g
- Housing: animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum
- Water: Animals received tap water, ad libitum
- Acclimatisation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 Feb 2016 To: 16 Mar 2016

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

50, 25 or 10% (w/v)

No. of animals per dose:

5

Details on study design:

ANIMAL ASSIGNMENT AND TREATMENT
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test substance, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test substance at a concentration of 50% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured, pre-dose on Day 1, post-dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25 % was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

DOSE SELECTION RATIONALE
Based on the results of the preliminary screening test, three groups of five mice were treated with the test substance at concentrations of 50%, 25% or 10% w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test substance would not produce systemic toxicity or excessive local irritation at the highest suitable concentration.  The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test substance formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the disposable pipette tip. A further group (vehicle control group) of five mice received the vehicle alone in the same manner. The positive control animals were similarly treated to the test animals except that 25 µL of the positive control substance, α Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

TREATMENT PREPARATION AND ADMINISTRATION
The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test substance formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of five mice received the vehicle alone in the same manner.

- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation.  For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
- Preparation of single cell suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was re suspended in 10 mL of PBS and repelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5 % Trichloroacetic acid (TCA).
- Determination of 3HTdR incorporation: After approximately eighteen hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by  scintillation counting. The vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system .

Statistics:

STATISTICS / DATA EVALUATION
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.

Probability values (p) are presented as follows:
-  P < 0.001 ***
-  P < 0.01 **
-  P < 0.05 *
-  P > 0.05 (not significant)

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test substance will be regarded as a sensitizer if at least one concentration of the test substance results in a threefold or greater increase in 3HTdR incorporation compared to control values.  Any test substance failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer." The EC3 value was also calculated. The EC3 value is the concentration of test substance expected to cause a 3-fold increase in 3HTdR incorporation.

Positive control results:

No clinical signs were noted in the positive control animals during the test. See Table 1 in 'Any other information on results incl. tables'.

Key result

Parameter:

EC3

Value:

41.2

Parameter:

SI

Value:

2.51

Test group / Remarks:

10 % (w/v)

Remarks on result:

other: negative result

Parameter:

SI

Value:

2.89

Test group / Remarks:

25 % (w/v)

Remarks on result:

other: negative result

Parameter:

SI

Value:

3.06

Test group / Remarks:

50 % (w/v)

Remarks on result:

other: positive result

Parameter:

SI

Value:

4.2

Test group / Remarks:

Positive Control Substance (α-Hexylcinnamaldehyde)

Remarks on result:

other: positive result

Cellular proliferation data / Observations:

PRELIMINARY TEST
Off white residual test item on the ears was noted on Days 2 and 3. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information, the test item at concentrations of 50%, 25% and 10% w/w in acetone/olive oil 4:1 were selected for the main test.

MAIN TEST
- One animal treated with the test substance at a concentration of 50 % w/w and one animal treated with the test substance at a concentration of 2 5% w/w were found dead on Day 4. No previous clinical signs had been noted in these animals and the deaths did not affect the purpose or integrity of the testing as an adequate number of animals remained to make an accurate assessment of skin sensitisation potential.
- No clinical signs were noted in the surviving test, vehicle or positive control animals during the test.
- Body weight change of the surviving test animals between Day 1 and Day 6 were comparable to that observed in the corresponding vehicle control group animals over the same period.

Table 1: Skin sensitisation potential of the test substance

Treatment Group	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle acetone/olive oil 4:1	1-1	317.21	370.71 (± 98.77)	NA	NA
	1-2	539.88
	1-3	307.53
	1-4	377.44
	1-5	311.50
Test substance 10% w/w in acetone/olive oil 4:1	2-1	1101.17	929.58** (± 252.87)	2.51	Negative
	2-2	1128.76
	2-3	1101.33
	2-4	729.02
	2-5	587.62
Test substance 25% w/w in acetone/olive oil 4:1	3-1	1162.12	1072.42#** (± 361.21)	2.89	Negative
	3-2	+
	3-3	1163.30
	3-4	1405.53
	3-5	558.51
Test substance 50% w/w in acetone/olive oil 4:1	4-1	1173.25	1134.85#*** (±70.36)	3.06	Positive
	4-2	1034.49
	4-3	1139.51
	4-4	1192.15
	4-5	+
Positive Control substance 25% v/v in acetone/olive oil 4:1	5-1	1652.87	1555.67*** (± 207.61)	4.20	Positive
	5-2	1793.61
	5-3	1656.39
	5-4	1313.09
	5-5	1362.41

#       Mean dpm/Animal obtained by dividing the dpm/Animal value by 8 (due to the death of one animal on Day 4).

**       Significantly different from control group p < 0.01

***       Significantly different from control group p < 0.001

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In conclusion, under the conditions of the present assay, the test substance, tested in a suitable vehicle (4:1 Acetone/Olive oil) was shown to have skin sensitization potential in the Local Lymph Node Assay.

Executive summary:

An OECD 429 study in compliance with GLP was performed to assess the skin sensitization potential of the test substance in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.  Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay (LLNA). Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test substance as a solution/emulsion in acetone/olive oil 4:1 at concentrations of 50 %, 25 % or 10 % w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone.  A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α Hexylcinnamaldehyde tech., 85 %, at a concentration of 25 % v/v in acetone/olive oil 4:1.    

Two animals treated with the test substance in the main test were found dead on Day 4 (one animal treated with 25 % w/w the test substance and one animal treated with 50 % w/w the test substance).  No other clinical signs were observed for the test item treated animals during the study, and there were no treatment related effects were observed on body weight. The Stimulation Index was expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group. The concentration of the test substance expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 41.2 %.

In conclusion, under the conditions of the present assay, the test substance, tested in a suitable vehicle (4:1 Acetone/Olive oil) was shown to have skin sensitization potential in the Local Lymph Node Assay.  

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation
The skin sensitization potential of the test substance in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear was studied under GLP to OECD TG 429. Following a preliminary screening test, in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose in the main test of the Local Lymph Node Assay (LLNA). Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test substance as a solution/emulsion in acetone/olive oil 4:1 at concentrations of 50%, 25% or 10% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer α Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1. Two animals treated with the test substance in the main test were found dead on Day 4 (one animal treated with 25% w/w the test substance and one animal treated with 50% w/w the test substance). No other clinical signs were observed for the test item treated animals during the study, and there were no treatment related effects on body weight. The Stimulation Index was expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group. The concentration of the test substance expected to cause a 3-fold increase in 3HTdR incorporation (EC3 value) was calculated to be 41.2%.

In conclusion, under the conditions of the present assay, the test substance tested in a suitable vehicle was shown to have skin sensitization potential in the Local Lymph Node Assay.  

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The test substance is not expected to be of concern for respiratory sensitisation. Following the recommended approach outlined in R.7.3.12.3, the test substance is not expected to be a respiratory sensitiser because the test substance is not di-isocyanate or protein. Furthermore, no structural warnings for respiratory sensitisation were found using the QSAR toolbox (v4.4).

Justification for classification or non-classification

Based on the available information the substance is classified as skin sensitiser category 1B, H317: May cause an allergic skin reaction in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.