2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Endpoint summary

Description of key information

An LLNA assay according to OECD 429 was positive for sensitization. The trio of non-animal studies (DPRA, Keratinosense, h-Clat) was initially conducted, but inconclusive results were obtained, resulting in the need for the LLNA.

The Direct Peptide Reactivity Assay or DPRA (OECD 442C) showed a negative result (“no to minimal” reactivity) and the KeratinoSensTM test method (OECD 442D) was also negative for MCS-1562. The result from the Human Cell Line Activation Test or h-CLAT (OECD 422E), however, was considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP). As the results were inconclusive, to determine the skin sensitizing potency and to conclude on classification of MCS-1562, a LLNA (OECD 429) study was performed. The LLNA reported MCS-1562 to be a Category 1B skin sensitizer.

This is documented in the attached document " MCS 1562 Sensitization Assessment". An assessment of the 3 in vitro studies is found in the "Skin sensitization in vitro data assessment" file.

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:

2018

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)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Identification: 2-ethylhexyl 7 -oxabicyclo [ 4 .1. 0 ]heptane-3-carboxylate
Batch: AH03201
CAS Number: 62256-00-2
Purity: 100%
Physical state I Appearance: Clear colourless liquid
Expiry Date: 01 February 2019
Storage Conditions: Approximately 4 °C in the dark

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

Animal Information

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

Animal Care and Husbandry

The animals were housed in suspended solid floor polypropylene cages furnished with softwood wood flakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

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

Concentration:

50 or 25%

No. of animals per dose:

4

Details on study design:

Test Item Administration
Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil4: 1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 11L of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item 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 four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL (250 11L) of phosphate buffered saline (PBS) containing 3H-methyl thymidine eHTdR: 80 11Ci/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 11Ci to each mouse.

Observations
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).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 cell 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 pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA). Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HT dR incorporation was measured by scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 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 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

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

Statistics:

The proliferation response of lymph node cells was expressed as the number of radioactive
disintegrations per minute per lymph node (disintegrations per minute/node) 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 item will be regarded as a sensitizer if at least one concentration of the test item
results in a threefold or greater increase in 3HTdR incorporation compared to control values.
Any test item 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 item expected
to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of
EC3 is:
EC3 = c + [[(3-d)/(b-d)] x (a-c)]

Major Computerized Systems
The following computerized system was used in the study:
Delta Controls- ORCA view

Positive control results:

Acceptible results were produced in the periodic positive control tests

Parameter:

EC3

Value:

33.6

Parameter:

SI

Value:

2.29

Test group / Remarks:

25%

Parameter:

SI

Value:

4.36

Test group / Remarks:

50%

Parameter:

SI

Value:

7.64

Test group / Remarks:

100%

Tables were not able to be pasted from a .PDF successfully, so they are attached as a single document attachment

Interpretation of results:

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

Conclusions:

The test item was considered to be a sensitizer under the conditions of the test

Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary  screening test in which no clinical signs of toxicity were noted at a concentration  of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.  Three groups, each of four animals, were treated with 50 ul (25 ul per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v.  A further group of four animals was treated with acetone/olive  oil 4:1 alone.

Results

The Stimulation  Index expressed as the mean radioactive incorporation  for each treatment group divided by the mean radioactive incorporation  of the vehicle control group are as follows:

Concentration (% v/v) in              Stimulation Index              Result

acetone/olive oi (4:1)

25                                                        2.29                     Negative

50                                                        4.36                     Positive

100                                                      7.64                     Positive

The concentration  of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 33.6%.

Conclusion

The test item was considered to be a sensitizer under the conditions of the test.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

The study was conducted in 1986, which was before the LLNA was available

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Hartley albino guinea pigs from Murphy Breeding Laboratories, Inc., were used in this study. They were ordered from the supplier in a weight range of 250 to 350 grams. Upon receipt in the laboratory the animals were maintained in quarantine for at least four days. Tap water and PURINA GUINEA PIG CHCW was available ad libitum throughout the study. Animals were housed singly in wire mesh suspension cages and were maintained on a 12-hour light/12-hour . dark cycle. Animals were identified by numbered cage cards. Careful checking of the cage cards and restrainers was used to assure the correct identity of each animal.

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

6 hours

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: acetone

Concentration / amount:

50%

Day(s)/duration:

6 hours

Adequacy of challenge:

not specified

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: Acetone

Concentration / amount:

50, 20 and 5% test article concentrations

Day(s)/duration:

6 hours

No. of animals per dose:

10/sex

Details on study design:

Primary irritation - Prior to induction and primary challenge, the test material was applied to guinea pigs to determine the concentrations appropriate for use during these study phases. For this purpose, MCS-1562 was tested undiluted and as 50%, 25%, and 10% w/v formulations in 95% ethanol. The test material was also tested as 50%, 25%, 10%, and 5.0% w/v formulations in acetone.

On the day before the applications were made, the backs of the animals were clipped with electric clippers to provide enough area to test four concentrations on each guinea pig. On the following day, the animals were placed in stainless steel restrainers and patches were applied to each animal. The patches, incorporating a 25 mm Hill Top Chamber, were moistened with 0.3 ml of one of the test formulations. The patches were occluded with dental dam placed over the back of each animal and secured with metal clips. The animals were restrained for six hours after Which the patches were removed and the animals returned to their cages.

On the day following applications, the clipped areas were depilated with NEET CREAM HAIR REMOVER (Whitehall Laboratories, Inc.). The depilatory was allowed to remain on the sites for eight minutes and then washed off with warm tap water. The patch sites were scored for severity of response at 24-hours and 48-hours without additional depilation, according to the following scale: 0 - no reaction; + ~ slight patchy erythema (Grades of + are equal to 0.5 for calculating the mean); 1 =slight confluent or moderate patchy erythema; 2 = moderate erythema and 3 • severe erythema with or without edema. The undiluted test material was chosen for use at induction, while a 50% w/V formulation in acetone was chosen for use at primary challenge. The test material at SO%, 20%, and 5.0% w/v formulations in acetone was chosen for use at rechallenge.

Induction of Sensitization - on the day prior to induction, the upper left quadrant of the backs of guinea pigs was clipped free of hair with electric clippers. On the following day chambers moistened with 0.3 rn1 of undiluted MCS-1562 were applied to 20 guinea pigs in the test group. Animals were restrained as previously described for six hours after which the patches were removed and the animals returned to their cages.

The chambers (moistened with 0.3 m1 of the undiluted material) were reapplied to the same site once each week for a total of three applications. The same site was clipped on the day before each application was made. The restraint periods lasted for six hours on each occasion.

Primary Challenge - Approximately two weeks after the last of the three induction applications, a fresh application site was prepared by clipping the lower left quadrant of the backs of the test and naive control animals. On the next day, December 16, 1986, a challenge patch moistened with 0.3 ml of MCS-1562 as a 50% w;v formulation in acetone was applied to each guinea pig in the test and naive control group. The animals were restrained as previously described for six hours after which the patches were removed and the animals returned to their cages.

On the day following the application the sites were depilated for eight to ten minutes and scored for severity of response 2 3/4 hours later (24-hour reading) by the methods previously described. The sites were scored again the next day for a 48-hour reading without additional depilation.

Scores of l or greater in the test group were considered to be indicative of sensitization providing grades of less than 1 were present in the naïve control group. If grades of 1 or greater were found in the naive control group, then reactions exceeding the most severe reactions in the control group were presumed to be due to sensitization.

Re-Challenge - Seven days following the primary challenge application, all of the original test animals and ten naive control animals were to be triple patch rechallenged, with MCS-1562 as 50%, 20%, and 5.0% w/v formulations in acetone. Due to a sample shortage only nine control animals were patched. Applications were as described in the Protocol Deviation section. The animals were restrained as previously described for six hours after which the patches were removed and the animals returned to their cages.

On the day following the application the sites were depilated for eight to eleven minutes and scored for severity of response 4 1/4 hours later (24-hour reading) by the methods previously described. The sites were scored again the next day for a 48-hour reading without additional depilation.

Challenge controls:

Yes

Positive control substance(s):

not specified

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

5%

No. with + reactions:

0

Total no. in group:

10

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

5%

No. with + reactions:

0

Total no. in group:

10

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

20%

No. with + reactions:

0

Total no. in group:

10

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

20%

No. with + reactions:

0

Total no. in group:

10

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Tables are attched as a combined .PDF file. IUCLID would not allow the tables to be pasted without losing fidelity.

Interpretation of results:

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

Conclusions:

The test article was sensitizing under the conditions of the study

Executive summary:

The potential of the test material, MCS-1562, to produce delayed contact hypersensitivity in guinea pigs was evaluated by a modification of the Buehler test.

 

The undiluted test material was applied to the backs of male and female Hartley guinea pigs for three six-hour periods at weekly intervals.

 

Group sizes were 20 animals for the test group and 10 animals for the naïve control group. Approximately two weeks after the last induction session, the test material prepared as a 50% w/v formulation in acetone was reapplied to the test animals for six hours. The test material was also applied to ten naive control animals. The next day the sites were depilated and scored for severity of response 24-hours and 48-hours after removal of the patches. 

 

The incidence of grade 1 responses in the test group (3 of 20) was compared to that of the naive control group, (0 of 10) at primary challenge. The incidence and severity of the responses in the test group was more pronounced than that produced by the naive control group suggesting that sensitization had been induced. Relative to the undiluted level of induction it could be said that this material induced weak sensitization.

 

Seven days following the primary challenge application, all of the original test animals and ten naive animals, were to be triple patch rechallenged, with MCS-1562 as 5O%, 20%, and 5.0% w/v formulations in acetone and 24- and 48-hour irritation scores obtained. Due to a sample shortage only nine control animals were patched.

 

The incidence of grade 1 responses in the rechallenge test group at the 50%, 20%, and 5.0% levels (7 of 20, 1 of 20, and 0 of 20, respectively) were compared to the naive control animals ( 0 of 8, 0 of 5, and 0 of 9, respectively). The incidence of grade+ responses in the rechallenge test group at the 5.0% level (11 of 20) was compared to the naive control group (1 of 9). The incidence and severity of the responses in the test group was more pronounced than that produced by the naive control group confirming that sensitization had been induced. In addition, the test animals exhibited responses characteristic of a dose response. Although no grades of 1 were exhibited at the 5.0% level the hyperreactivity of + grades suggests that a no effect level was not achieved.

Reference 3

Endpoint:

skin sensitisation, other

Type of information:

other: Skin sensitization in vitro data assessment

Adequacy of study:

other information

Executive summary:

The testing laboratory conduted a data assessment of all 3 in vitro studies. The assessment is attached here

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

An LLNA assay according to OECD 429 was positive for sensitization. The trio of non-animal studie (DPRA, Keratinosense, h-Clat) was initially conducted, but inconclusive results were obtained, resulting in the need for the LLNA. This is documented in the attached document "Skin Sensitization In Vitro study data assessment...... summary"

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