Dapsone

Endpoint summary

Description of key information

Assessment of contact hypersensitivity to 4,4'-DDS in the mouse (Local Lymph Node Assay, 5 females/dose) was conducted according to OECD 429 guidelines and GLP principles.

Based on its results, 4,4’-DDS would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.

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:

August 03 -22, 2011

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)

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 (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Number of animals: 20 females (nulliparous and non-pregnant), five females per group.
- Age at study initiation: Young adult animals (approx. 11 weeks old)
- Weight at study initiation: 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 (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Health inspection: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 – 23.8
- Humidity (%): 40 - 70
Due to a technical failure of the temperature and relative humidity sensors of the study room after 05 July 2011 (10:43), temperature and relative humidity data were used from an adjacent room connected to the same temperature and relative humidity regulation system as the study room. Temperature and relative humidity inside this room was therefore considered representative for these conditions in the study room.
Deviations from the maximum level of relative humidity occurred.Evaluation: Laboratory historical data do not indicate an effect of the deviations.
The study integrity was not adversely affected by the deviation.
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 03, 2011 to August 22, 2011

Vehicle:

dimethyl sulphoxide

Concentration:

Pre-screen test: a 25% and 50% concentration
Main study: 0, 10, 25 and 50% w/w

No. of animals per dose:

5

Details on study design:

The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give moderate irritation at the most (maximum grade 2 (see section 6.7) and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.

The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 12.5 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.

Animals were sacrificed after the final observation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance 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 (2007) 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.
Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3)

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: 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.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

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 stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that the vehicle was administered instead of the test substance.

Excision of the nodes - Day 6:
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 (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, 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 μm). 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) 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 (PerkinElmer 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 came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
0: No erythema
1: Very slight erythema (barely perceptible)
2: Well-defined erythema
3: Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth)
4: Severe erythema (beet redness) to eschar formation preventing grading of erythema

Oedema formation:
0: No oedema
1: Slight oedema (barely perceptible)
2: Moderate oedema
3: Severe oedema

Necropsy: All animals were subjected to necropsy for gross macroscopic examination.

Positive control substance(s):

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

Statistics:

Not performed.

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See Appendix I 'Reliability check' in the attached document.

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

The SI values calculated for the substance concentrations 10 and 25% were 1.5 and 1.4 respectively.

Key result

Parameter:

other: disintegrations per minute (DPM)

Value:

666

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 988, 949 and 2256 DPM respectively. The mean DPM/animal value for the vehicle control group was 666 DPM.

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

The SI values calculated for the substance concentrations 10 and 25% were 1.5 and 1.4 respectively.

Tables and figures of the Pre-screen test and Main study have been included in the attached document "LLNA tables and figures".

Results Pre-screen test:

No irritation was observed in any of the animals examined. Notable body weight loss was noted for both animals at 25%, but was less pronounced or within normal ranges for the animals at 50%. No clinical signs of systemic toxicity were observed in any of the animals. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. White test substance remnants were present on the dorsal surface of the ears of both animals at 50% (Days 3 to 5), which did not hamper scoring of the skin reactions.

 

Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

Other results - main study:

No irritation of the ears was observed in any of the animals examined.

White test substance remnants were present on the dorsal surface of the ears of all animals at 50% (Days 2 to 5), which did not hamper scoring of the skin reactions.

 

One or both auricular lymph nodes of three animals at 50% appeared larger in size when compared to the other treated groups. All other auricular lymph nodes were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

One animal at 50% (no. 18) was found dead on Day 4. Another animal at 50% (no. 20) was sacrificed on Day 4 for ethical reasons due to a missing right ear. Restless behaviour was noted for all animals at 10% on Days 2 and 3, at 25% between Days 2 and 4, and at 50% between Days 2 and 6. All animals at 25 and 50% also showed hunched posture on Day 4. The animal at 50% found dead on Day 4 also had a missing left ear and showed piloerection on Day 4. No mortality occurred at 10 and 25%.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Interpretation of results:

GHS criteria not met

Conclusions:

Assessment of Contact Hypersensitivity to 4,4'-DDS in the Mouse (Local Lymph Node Assay, 5 females/dose) was conducted according to OECD 429 guidelines and GLP principles.

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 25%, 4,4’-DDS was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%.

It does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Executive summary:

Assessment of Contact Hypersensitivity to 4,4’-DDS in the Mouse (Local Lymph Node Assay) was carried out based on the guidelines OECD, Section 4, Health Effects, No.429 (2010) and EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay". Test substance concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w 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 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.

No irritation of the ears was observed in any of the animals examined. White test substance remnants were present on the dorsal surface of the ears of all animals at 50% (Days 2 to 5), which did not hamper scoring of the skin reactions. One animal at 50% (no. 18) was found dead on Day 4. Another animal at 50% (no. 20) was sacrificed on Day 4 for ethical reasons due to a missing right ear. Restless behaviour was noted for all animals at 10% on Days 2 and 3, at 25% between Days 2 and 4, and at 50% between Days 2 and 6. All animals at 25 and 50% also showed hunched posture on Day 4. The animal at 50% found dead on Day 4 also had a missing left ear and showed piloerection on Day 4. No mortality occurred at 10 and 25%. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

One or both auricular lymph nodes of three animals at 50% appeared larger in size when compared to the other treated groups. All other auricular lymph nodes were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 988, 949 and 2256 DPM respectively. The mean DPM/animal value for the vehicle control group was 666 DPM.

At 50%, clinical signs of systemic toxicity were observed among all animals, and two animals did not survive until scheduled necropsy. Therefore, the 50% concentration was not used for interpretation and no SI value was calculated. The SI values calculated for the substance concentrations 10 and 25% were 1.5 and 1.4 respectively.

At 10 and 25%, all animals showed restless behavior, and the SI value at these lower concentrations was clearly below 3. Given the nature of this clinical appearance, it was considered most likely that concentrations below 10% - had these been tested - would also have resulted in an SI below 3 and would therefore not have influenced the conclusion of this study.

 

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 25%, 4,4’-DDS was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%.

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity

Based on these results, 4,4’-DDS would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Assessment of contact hypersensitivity to 4,4'-DDS in the mouse (Local Lymph Node Assay, 5 females/dose) was conducted according to OECD 429 guidelines and GLP principles. Based on its results, 4,4’-DDS would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.