Reaction mass of 604-916-7 and (4R,4aS,5aR,12aS)- 4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-9-nitro-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide sulfate (1:2),

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 May 2011 to 23 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No.429, "Skin Sensitization: Local Lymph Node Assay", Paris Cedex, July 2010.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B42: "Skin Sensitization: Local Lymph Node Assay". Official Journal of the European Union No. L142, May 2008, including most recent amendments.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.2600. “Skin Sensitization”, March 2003.

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(4S,4aS,5aR,12aS)- 4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-9-nitro-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide sulfate (1:2)

EC Number:

604-916-7

Cas Number:

153621-80-8

Molecular formula:

C23H30N4O17S2

IUPAC Name:

(4S,4aS,5aR,12aS)- 4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-9-nitro-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide sulfate (1:2)

Test material form:

solid: particulate/powder

Details on test material:

Purity 80-100%

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

6.2. Test system
Species Mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).

Source: Janvier, Le Genest-Saint-Isle, France

Number of animals 20 females (nulliparous and non-pregnant), five females per group.

Age and body weight Young adult animals (approx. 9 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.

Identification Tail mark with marker pen.

Health inspection A health inspection was performed prior to treatment, to ensure that the animals were in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

Reliability check The results of a reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures is summarized in the appendix of this report. For both scientific and animal welfare reasons, no concurrent positive control group was added to the study.
An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.

6.3. Animal husbandry
Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 21.0 - 23.6ºC), a relative humidity of 40-70% (actual range: 35 - 70%) and 12 hours artificial fluorescent light and 12 hours darkness per day.

Accommodation
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) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment. Certificates of analysis were examined and then retained in the NOTOX archives.

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
Free access to tap water.

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.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

10, 25, 50 %(w/w)

No. of animals per dose:

5 females

Details on study design:

6.6. Main study
Three groups of five animals were treated with one test substance concentration per group. The
highest test substance concentration was selected from the pre-screen test.
One group of five animals was treated with vehicle.

6.6.1. Allocation
Group animal numbers induction (test substance; % w/w)
1 01 - 05 0 (Dimethyl formamide )
2 06 - 10 10
3 11 - 15 25
4 16 - 20 50

6.6.2. 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 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 substance.

6.6.3. 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 (0.2 mL/animal) 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.

6.6.4. 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.

6.6.5. 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):

other: concurrent vehicle

Statistics:

6.9. Interpretation
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, based on the test guideline and recommendations done by ICCVAM (Ref. 1).

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) (Ref. 2).

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

ca. 6.5

Variability:

± 1.7

Test group / Remarks:

10% Concentration

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Results 2

Parameter:

SI

Value:

ca. 10.7

Variability:

± 2.8

Test group / Remarks:

25% Concentration

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Results 3

Parameter:

SI

Value:

ca. 5.6

Variability:

± 1.2

Test group / Remarks:

50% Concentration

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Results 4

Parameter:

EC3

Value:

>= 0 - <= 10

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Cellular proliferation data / Observations:

7.1. Pre-screen test (Table 1, Table 2 and Table 3)
Brown remnants of the test substance were present on both ears of all animals at 25 and 50% between Days 1 and 6, due to which scoring for erythema was not possible between Days 1 and 4. No signs of necrosis or oedema were present, and no signs of systemic toxicity were noted at a 25 and 50% concentration. Bald areas behind the ears were noted for both animals at 50% between Days 3 and 6. An increase in ear thickness exceeding 25% from Day 1 pre-dose values was observed at a 50% concentration for one animal. Variations in ear thickness for the other animal at a 50% concentration and for both animals at a 25% concentration were less than 25% from Day 1 pre-dose values.

Since it could not be excluded that the increase in ear thickness was due to test substance remnants, and since there were no signs of irritation, the highest test substance concentration selected for the main study was a 50% concentration. Ear thickness measurements were also included for the 50% concentration in the Main study to support this selected highest concentration.

7.2. Main study
7.2.1. Skin reactions / Irritation (Table 4: Body weights and erythema and Table 5)
Brown remnants of the test substance were present on both ears of all animals at 10, 25 and 50% between Days 1 and 6, due to which scoring for erythema was not possible between Days 1 and 4 (all concentrations) and for some animals at a 10 and 25% concentration on Day 5. Bald areas behind the ears were noted for all animals at 50% between Days 3 and 6. Crusts were present behind the ears of all animals at 50% and for one animal at 10% on Days 5 and 6. It was considered that these crusts represented test substance remnants, since the observed test substance remnants had a brown appearance, and there were no signs of excessive irritation among any animal up to the highest concentration tested.

No oedema was observed in any animal. The very slight irritation of the ears as shown by one animal at 10% and all animals at 50% was considered not to have a toxicologically significant effect on the activity of the nodes. Also, variations in ear thickness at a 50% concentration were less than 25% from Day 1 pre-dose values, which indicated that this concentration was suitable for use in the main study.

7.2.2. Body weights (Table 4)
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 a few animals, was considered not toxicologically significant.

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

7.2.4. Macroscopy of the auricular lymph nodes and surrounding area (Table 6)
All auricular lymph nodes at 25 and 50% and the majority of auricular lymph nodes at 10% appeared larger in size when compared to the control group. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

7.2.5. Radioactivity measurements (Table 6)
Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 2164, 3571 and 1860 DPM respectively. The mean DPM/animal value for the vehicle control group was 334 DPM.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on these results:
- according to the recommendations made in the test guidelines, 9-Nitrominocycline would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007), 9-Nitrominocycline should be classified as skin sensitizer (Category
1).