1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

Endpoint summary

Description of key information

Skin sensitisation:

No suitable solvent is available to test T002632 for skin sensitizing properties in the KeratinoSens ^TM and U-SENS ^TM assays and

therefore performance of these assays was technically not feasible. As a result, the in vitro skin sensitization testing strategy was not initiated, as insufficient information to conclude on skin sensitizing properties would be acquired from the outcome of the

in chemico test (DPRA) and DEREK result.

In silico: A Derek Nexus assessment yielded an alert for skin sensitisation based on the presence of an Alkyl aldehyde precursor (Alert 424). and predicted an EC3 of 44%.

In vivo: The results of the Local Lymph Node Assay (van Sas, P., 2019, K1, OECD 429) showed that the test item was regarded as skin sensitizer (Category 1B).

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:

2019-01-23 to 2019-04-01

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Official Journal of the European Union No. L142, May 2008, including most recent amendments

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I18IB2522
- Expiration date of the lot/batch: 2020-03-17
- Purity/composition correction factor: correction factor is 1.00
- Purity: 100.1% w/w GC

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle:
Vehicle: Propylene glycol
Solubility in Propylene glycol : Not indicated
Stability in Propylene glycol : Stable for 6 hours

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item was not applicable, since the test method required a logical concentration range rather than specific dose levels to be dosed.

Species:

mouse

Strain:

CBA:J

Remarks:

inbred, SPF-quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12 weeks old
- Weight at study initiation: 18.9 to 25.4g. Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material. Paper and shelters (disposable paper corner home) were supplied as cage-enrichment.
- Diet (e.g. ad libitum): ad libitum pelleted rodent diet
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions
- Indication of any skin lesions: It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 2019-01-23 to 2019-04-01

Vehicle:

propylene glycol

Concentration:

Pre-screen test: 25% and 50%
Main study: 0% (propylene glycol), 2%, 10%, 25%

No. of animals per dose:

Five females per group; 4 groups (including control group)

Details on study design:

Rationale vehicle selection:
The vehicle was selected based on the stability data obtained in the Test Facility Study 20161739. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol and dimethylsulfoxide.

PRE-SCREEN TESTS:
- At a 50% test item concentration hunched posture was noted on Day 2 and/or 3. At a 25% test item concentration no signs of systemic toxicity were noted and up to very slight irritation was observed.
- At 25% and 50%, variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.
- White test item remnants were present on the dorsal surface of the ears of the animals at 25 and 50% on Days 2 and/or 3, which did not hamper scoring of the skin reactions.
- Based on these results, the highest test item concentration selected for the main study was a 25% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
- Criteria used to consider a positive response:
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, including all amendments. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3).

Classification of results:
SI value UN-GHS 2017; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer -
SI ≥ 3 Cat 1 Skin sensitizer H317: May cause an allergic skinreaction
EC3 value ≤ 2%: sub-category 1A
EC3 value > 2%: sub-category 1B

TREATMENT PREPARATION AND ADMINISTRATION
Induction - Days 1, 2 and 3: The dorsal surface of both ears was topically treated (25 μL/ear or an equivalent amount when dosed with a spatula) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the nodes - Day 6: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine.
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity measurements - Day 7: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

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

Statistics:

No data

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used bythe laboratory. In this study, performed in January 2019, females of the CBA/J mouse strain were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade (HCA). The females were approximately 11 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKCD3159. Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v; AcOO).
The SI values calculated for the item concentrations 5, 10 and 25% were 1.3, 1.4 and 5.2, respectively.
An EC3 value of 16.3% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8, 19.2 and 14.3%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at the laboratory is an appropriate model for testing for contact hypersensitivity.

Parameter:

SI

Value:

0

Variability:

+/- 0.2

Test group / Remarks:

Based on 5 animals in the control group

Parameter:

SI

Value:

2.7

Variability:

+/- 0.5

Test group / Remarks:

Based on 5 animals in the 2% w/w group

Parameter:

SI

Value:

4.6

Variability:

+/- 0.6

Test group / Remarks:

Based on 5 animals in the 10% w/w group

Parameter:

SI

Value:

5.4

Variability:

+/- 0.8

Test group / Remarks:

Based on 5 animals in the 25% w/w group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
mean DPM ± SEM:
0% w/w group: mean DPM ± SEM: 953 ± 169
2% w/w group: mean DPM ± SEM: 2548 ± 485
10% w/w group: mean DPM ± SEM: 4430 ± 607
25% w/w group: mean DPM ± SEM: 5166 ± 799
DPM = Disintegrations per minute
SEM = Standard Error of the Mean

EC3 CALCULATION
These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 3.3% was calculated.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: The very slight erythema of the ears as shown by animals treated at 10% and 25% was considered not to have a toxicologically significant effect on the activity of the nodes.
- Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- Macroscopy of the auricular lymph nodes and surrounding area: All auricular lymph nodes were considered normal in size, except for one node in a single animal treated at a concentration of 10%. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Radioactivity measurements and SI values: Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 10 and 25% were 2548, 4430 and 5166 DPM, respectively. The mean DPM/animal value for the vehicle control group was 953 DPM. The SI values calculated for the item concentrations 2, 10 and 25% were 2.7, 4.6 and 5.4, respectively.

Interpretation of results:

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

Conclusions:

These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 3.3% was calculated.

Based on these results:
- According to the recommendations made in the test guidelines (including all amendments), JNJ-26144612-AAA (T002632) would be regarded as skin sensitizer.
- According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), JNJ26144612-AAA (T002632) should be classified as skin sensitizer (Category 1B).
- According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), JNJ26144612-AAA (T002632) should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

Reference 2

Endpoint:

skin sensitisation: in chemico

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 3

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In accordance with point 8.3.2 of Annex VII of REACH, if in vitro/in chemico test methods are not applicable or adequate for classification and risk assessment of the substance, an in vivo test may be performed. Since it is concluded that it is not possible to address this endpoint by a non-animal testing strategy, it was considered justified to perform an in vivo study to assess and potentially classify the skin sensitizing properties of JNJ-26144612-AAA (T002632).

The rationale for not performing in vitro studies is described in detail in the Weight-of-Evidence justification attached to this endpoint summary.

In the main study of the loval lymph node assay (van Sas, P., 2019, K1, OECD 429), three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Propylene glycol).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes were considered normal in size, except for one node in a single animal treated at a concentration of 10%. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 10 and 25% were 2548, 4430 and 5166 DPM, respectively. The mean DPM/animal value for the vehicle control group was 953 DPM. The SI values calculated for the test item concentrations 2, 10 and 25% were 2.7, 4.6 and 5.4 respectively.

These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 3.3% was calculated.

The test item was concluded to be a skin sensitizer (cat. 1B).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Taking into account the in vivo testing (Local Lymph Node Assay (LLNA) in mice according to OECD 429) has been performed to assess the potency of the test item. Based on the EC3 value calculated in the LLNA, according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) the test item should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.