Rifamycin, 3-amino-1,4-dideoxy-1,4-dihydro-1,4-dioxo-(9Cl)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-12-01 to 2018-01-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Rifamycin, 3-amino-1,4-dideoxy-1,4-dihydro-1,4-dioxo-

Cas Number:

51756-80-0

Molecular formula:

C37 H46 N2 O12

IUPAC Name:

Rifamycin, 3-amino-1,4-dideoxy-1,4-dihydro-1,4-dioxo-

Specific details on test material used for the study:

Batch No.: 17003R86A
Purity: ≥98%

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

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: Young adult animals (approximately 11 weeks old)
- Weight at study initiation: 19.3 to 25.0 g
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilized sawdust as bedding equipped with water bottles. Animals were separated during designated procedures/activities. Each cage was clearly labeled.
- Diet (e.g. ad libitum): Pelleted rodent diet was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap-water was freely available to each animal via water bottles.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures is 18 to 24°C, the actual is 21 to 22°C
- Humidity: Relative target humidity is 40 to 70%, the actucal is 42 to 45%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Pre-screen Test: first: 25% and 50% w/w, then 10% and 25% w/w
Main Test: 0, 2, 5 and 10 % (w/w)

No. of animals per dose:

Pre-screen Test: two animals per concentration
Main Test: five animals

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 50% concentration: the highest concentration that could be prepared homogeneously
- Results:
At a 50% and 25% test item concentration, very slight erythema and no signs of toxicity were noted. Variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and therefore this concentration did not meet the selection criteria.
At a 10% test item concentration, no signs of systemic toxicity were noted and no irritation was observed and therefore this concentration was selected as highest concentration for the main study.

MAIN STUDY:Three groups of five animals were treated with one test item concentration per group. One group of five animals was treated with the vehicle.
- Allocation/concetration: 0, 2, 5 and 10%
- Induction - Days 1, 2 and 3: The dorsal surface of both ears was topically treated (25 µL/ear 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 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) 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 (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).

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

the test item concentrations 2%

Results 2

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

the test item concentrations 5 %

Results 3

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

the test item concentrations 10 %

Cellular proliferation data / Observations:

- Skin Reactions / Irritation:
No irritation was observed in any of the animals.
Brown test item remnants were present on the dorsal surface of the ears of all test item treated animals between Days 1 and 3, which did not hamper scoring of the skin reactions.
- 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.
- Macroscopic Examination of the Lymph Nodes and Surrounding Area:
The majority of auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for one node of one animal treated at 2% which was considered to be reduced in size.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Radioactivity Measurements:
Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 375, 423 and 377 DPM, respectively. The mean DPM/animal value for the vehicle control group was 411 DPM.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.
Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.

Executive summary:

The objective of this study was to evaluate whether the test item induces skin sensitization in mice (Local Lymph Node Assay) after three epidermal exposures of the animals according to OECD 429. 

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone. 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 was observed in any of the animals.

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.

The majority of auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for one node of one animal treated at 2% which was considered to be reduced in size. 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, 5 and 10% were 375, 423 and 377 DPM, respectively. The mean DPM/animal value for the vehicle control group was 411 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 0.9, 1.0 and 0.9, respectively.

Since there was no indication that the test item elicits a SI≥3 when tested up to 10%, test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.

Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.