2-amino-5-bromopyridin-3-ol

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:

From 06 to 26 Jul 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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: Charles River France, L'Arbresle Cedex, France.
- Age at study initiation: Approx. 11 weeks old.
- Weight at study initiation: Body weight variation was within ±20% of the sex mean.
- Housing: Individual housing in labeled Macrolon cages containing sterilized sawdust as bedding material.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- 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±3.0℃ (actual range: 21.0-23.4℃ )
- Humidity (%): 40-70% (actual range: 40-78%)
- Air changes (per hr): Approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From: 2010-07-06 To: 2010-07-26

Vehicle:

dimethyl sulphoxide

Concentration:

In preliminary study: 25 and 50% w/w
In main study: 0, 10, 25 and 50% w/w

No. of animals per dose:

In preliminary study:
Two groups of one animal were treated with one test substance concentration per group.
In main study:
One group of five animals was treated with vehicle.
Three groups of five animals were treated with one test substance concerntration per group.

Details on study design:

Preliminary irritation study
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2) at the highest concentration.
Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration that could technically be applied. The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (in the range of 8 to14 weeks old, Source: Harlan, Horst, The Netherlands). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

Main study
1) Allocation
Three groups of five animals were treated with one test substance concentration per group (10, 25 and 50% w/w). One group of five animals was treated with vehicle.
2) Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 µL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were mixed thoroughly using a vortex mixer 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 substance.
3) Excision of the nodes - 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 (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.
4) 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) and stored in the refrigerator until the next day.
5) 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).

Positive control substance(s):

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

Statistics:

None stated

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The SI value for the vehicle control group was 1.0. The SI value for the experimental group treated with test substance concentrations 10% was 4.0. The SI value for the experimental group treated with test substance concentrations 25% was 7.7. The SI value for the experimental group treated with test substance concentrations 50% was 7.4.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The mean DPM/animal value for the vehicle control group was 660 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 10% was 2636 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 25% was 5100 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 50% was 4851 DPM.

No irritation of the ears was observed in the animals examined, except for one animal at 25% which showed slight erythma on one ear. This slight irritation was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined. Brown test substance remants were noted on the dorsal surface of the ears of animals at 50%, which did not hamper scoring for irritation.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Disintegrations Per Minute (DPM) and Stimulation Index (SI)

Group	Test substance (% w/w)	Mean
		DPM	SI
2	10%	2636	4.0
3	25%	5100	7.7
4	50%	4851	7.4
1	0% (vehicle)	660	1.0

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It was concluded that the test substance elicited SI values of >3 and hence is considered as a skin sensitiser under this studies conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A LLNA study was conducted according to OECD 429 using mouse (Beerens - Heijnen, 2010). Key study. This study indicate that the test substance could elicit an SI ≥ 3, hence the test substance should be classfied as a skin sensitizer.

Migrated from Short description of key information:
A LLNA (Beerens - Heijnen, 2010) study was available which is key study. This study showed that the test substance is skin sensitising.

Justification for selection of skin sensitisation endpoint:
This study was conducted according to OECD 429 under GLP.

Justification for classification or non-classification

Skin sensitisation: Animal tests gave positive results (LLNA SI ≥ 3 (actual value 7.7)).

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.4.2 the substance is classified as a skin sensitizer (Category 1).