2-{2-[(2R,4R,6R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl]ethyl}-1H-isoindole-1,3(2H)-dione

Endpoint summary

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:

09 January 2008 - 21 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed to GLP and according to appropriate OECD, EPA, and EC test guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L'Arbesle Cedex, France
- Age at study initiation: approximately 12 weeks old.
- Weight at study initiation: 20 - 25 g (Taken from Table 2 of the test report, Day 1 weights)
- Housing:Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezlaldiaten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0ºC (Actual range 19.5 - 23.7ºC).
- Humidity (%): 30 - 70% (actual range 42 - 68%).
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.


IN-LIFE DATES: From: 09 January 2008 To: 21 January 2008

Vehicle:

dimethyl sulphoxide

Concentration:

Induction concentrations, displayed as % test substance in DMSO;
Group 1 - 0 % (Diemethylsulfoxide, vehicle control)
Group 2 - 10 %
Group 3 - 25%
Group 4 - 50%

No. of animals per dose:

Five females in each group (dose)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Soluble in the vehicle (Dimethylsulphoxide).
- Irritation: Two animals were exposed to the test substance for three days, according to the exposure procedure used for days 1 - 3 of the main test. Animals were dosed at 25% and 50% test substance in DMSO; neither concentration induced a skin reaction so the highest concentration (50%) was used as the highest level in the main test.
- Lymph node proliferation response: Not assessed.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A Stimulation Index (SI) is calculated for each group; the SI is the ratio of the DPM for the test group / the DPM for the vehicle control group (refer below for details regarding DPM). If the results indicate an SI ≥ 3, the test substance may be regarded as a skin sensitizer.


TREATMENT PREPARATION AND ADMINISTRATION:
The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the
vehicle. Homogeneity was obtained to visually acceptable levels.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing.
The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.

Treatment - Day 6
All animals:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 µCi of 3H-methyl thymidine (GE Health care, Buckinghamshire, UK).
After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital Euthesate® (0.2 ml/animal) (Ceva Sante Animale BV, Naaldwijk, The Netherlands). 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
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 11m). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4° C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) at 4° C during the night.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (Perkin Elmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ±0.2% or a maximum of 5 minutes whichever comes 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):

not specified

Statistics:

N/A - Data were assessed as detailed above; no formal statistical analysis was performed.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Refer to table 2, below. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 2.0 and 4.5 respectively. These results indicate that the test substance could elicit an SI≥3. The data showed a dose response and an EC3 value of 35% was calculated.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Refer to table 1, below. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 691, 1293 and 2871 respectively. The mean DPM/animal value for the vehicle control group was 634

Table 1: Radioactivity measurements (individual animals)

Group	%Test substance ^1	Animal	DPM /animal
1	0% (Vehicle)	1	840
		2	295
		3	491
		4	672
		5	870
2	10%	6	823
		7	751
		8	675
		9	712
		10	493
3	25%	11	1049
		12	1573
		13	1530
		14	876
		15	1438
4	50%	16	3956
		17	2119
		18	969
		19	3680
		20	3630

¹Vehicle: DMSO

Table 2: Disintegrations Per Minute (DPM) and Stimulation Index (SI).

Group	% Test Substance^1	Mean DPM ± SEM	SI ± SEM
2	10%	691 ± 55	1.1 ± 0.2
3	25%	1293 ± 139	2.0 ± 0.4
4	50%	2871 ± 574	4.5 ± 1.2
1	0% (Vehicle)	634 ± 108	1.0

¹Vehicle: DMSO

Refer also to Attachment 1 for Report results tables.

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 2.0 and 4.5 respectively.
These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose response and an EC3 value of 35% was calculated.
Based on these results and according to the recommendations made in the test guidelines, LACTOL would be regarded as a skin sensitizer.

Executive summary:

A Local Lymph Node Assay (LLNA) was performed by NOTOX B.V., Netherlands, on behalf of Pfizer Ireland Phamaceuticals Ltd to assess the potential for skin sensitization of the test substance Lactol. The study was performed according to GLP and in accordance with OECD 492, EU Method B42 and to EPA Test Guidelines. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, three groups of five experimental animals were treated with test substance concentrations of 10%, 25% or 50% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Dimethyl sulphoxide). 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. 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. No irritation was observed in any of the animals examined and the majority of nodes were considered normal in size, except for the nodes of three animals of the highest dose group.

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 2.0 and 4.5 respectively. These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose response and an EC3 value of 35% was calculated.

Based on these results and according to the recommendations made in the Test Guidelines, LACTOL would be regarded as a skin sensitizer and should be assigned the symbol 'Xi' and the the risk phrase R43 - 'may cause sensitisation by skin contact'.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A Local Lymph Node Assay to assess the potential for skin sensitization of the test substance. The study was performed according to GLP and in accordance with OECD 492, EU Method B42 and to EPA Test Guidelines. No irritation was observed in any of the animals examined and the majority of nodes were considered normal in size, except for the nodes of three animals of the highest dose group. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 2.0 and 4.5 respectively. These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose response and an EC3 value of 35% was calculated.

Migrated from Short description of key information:

A Local Lymph Node Assay to assess the potential for skin sensitization of the test substance. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 2.0 and 4.5 respectively. These results indicate that the test substance could elicit an SI ≥ 3.

Justification for selection of skin sensitisation endpoint:

Study was performed to GLP and according to appropriate OECD, EPA, and EC test guidelines.

Justification for classification or non-classification

Based on the results of the test and according to the recommendations made in the Test Guidelines, the test substance would be regarded as a skin sensitizer and should be assigned the symbol 'Xi' and the the risk phrase R43 - 'may cause sensitisation by skin contact'.