Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
06 July 2010 - 19 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidlines and according to GLP principles
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
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 (CBA/J mice)
- Source: Harlan, Horst, The Netherlands,
- Age at study initiation: Young adult animals (approx. 10 weeks old) were selected
- Weight at study initiation: 21-24 gram. Body weight variation was within +/- 20% of the sex mean.
- 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.
During the acclimation period the accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).
- 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: At least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).
- 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 – 23.2ºC
- Humidity (%): 43 - 81%. Temporary deviations from the maximum level of relative humidity (70%) occurred. Laboratory historical data do not indicate an effect of the deviations.
- 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: 06 July 2010 To: 19 July 2010
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0-50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A 50% suspension of the test substance in Acetone/Olive oil (4:1 v/v) was selected as suitable formulation based on trial formulation results. Lower concentrations with this vehicle (up to 5%) or use of other vehicles up to a 10% concentration (eg. N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide or toluene/olive oil (2:1 v/v)) did not yield a homogenous formulation, or a brownish colour of the formulation indicated that oxidation had occurred. Water-based vehicles such as ethanol/water (7:3 v/v) and 1% pluronic L92 could not be used since the test substance should not come in contact with water.

- Irritation: A preliminary irritation study was conducted with a 50% concentration, which was the highest test concentration to be used in the main study. The selected concentration should be well tolerated systemically but may elicit moderate irritation (maximally grade 2) at the highest concentration.
The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study. One young adult animal was selected (11 weeks old). The animal was treated with a 50% concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweight was determined on Day 3. The animal was sacrificed after the final observation and no necropsy was performed.
-Lymp node proliferation response: Not determined in the preliminary irritation study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Mouse LLNA (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, based on the test guideline and recommendations done by ICCVAM.
The results were evaluated according to the Globally Harmonized System of Classification and Labeling 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, labeling 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:
The test substance was lyophilized, ground to a fine powder using an automatic grinder and stored under nitrogen in a dessicator (in order to avoid exposure to moist as much as possible). The white 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.

One group of five animals was treated with one test substance concentration. One group of five animals was treated with vehicle.

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 formulations were mixed thoroughly using a vortex mixer 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
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.

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.

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.
Toxicity: At least once daily.
Body weights: On Days 1 (pre-treatment) and 6.
Necropsy: No necropsy was performed according to protocol.
Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the modified Draize scoring system and descriptions of all other (local) effects were recorded.
Positive control substance(s):
other: The results of a reliability test with Alpha-hexylcinnamicaldehyde, performed not more than 6 months previously, are summarized in the appendix. Similar procedures were used in the reliability test and in this study.
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 attached document "Reliability check:.
Parameter:
SI
Remarks on result:
other: The SI value calculated for the substance concentrations 50% was 1.0.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The mean DPM/animal value for the experimental group treated with test substance concentrations 50% was 1052 DPM and the mean DPM/animal value for the vehicle control group was 1033 DPM.

Preliminary irritation study:

The results of the epidermal exposures for the 50% test substance concentration to be tested in the main study are presented in table 1.

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

Main study:

The slight erythema of the ears as shown by the animals treated at 50% 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.

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.

The mean DPM/animal value for the experimental group treated with test substance concentrations 50% was 1052 DPM and the mean DPM/animal value for the vehicle control group was 1033 DPM.

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

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Since there was no indication that the test substance elicits an SI ≥ 3 when tested at 50%, 202140/B was considered not to be a skin sensitizer. No EC3 value (the estimated test substance concentration that will give a SI =3) (if any) could be established.
Based on these results, 202140/B would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. It does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures. based on read across from ferrous sulphate, iron trinitrate also does not require classification as a potential skin sensitiser.
Executive summary:

The murine local lymph node assay was completed according to standard test guideline methods. The lab confirmed the validity of the methods with periodic positive control studies. Ferrous sulphate was administered in acetone:olive oil 4:1 v/v at three concentrations up to 50%.

The slight erythema of the ears as shown by the animals treated at 50% 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.

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.

The mean DPM/animal value for the experimental group treated with test substance concentrations 50% was 1052 DPM and the mean DPM/animal value for the vehicle control group was 1033 DPM.

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

Since there was no indication that the test substance elicits an SI ≥ 3 when tested at 50%, 202140/B was considered not to be a skin sensitizer. No EC3 value (the estimated test substance concentration that will give a SI =3) (if any) could be established. Based on these results, 202140/B would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. It does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures. based on read across from ferrous sulphate, iron trinitrate also does not require classification as a potential skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A murine local lymph node assay was completed according to standard test guideline methods and the validity of the methods was confirmed by periodic positive control studies. Ferrous sulphate was administered in acetone:olive oil 4:1 v/v at three concentrations up to 50%. The slight erythema of the ears as shown by the animals treated at 50% 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. 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. The mean DPM/animal value for the experimental group treated with test substance concentrations 50% was 1052 DPM and the mean DPM/animal value for the vehicle control group was 1033 DPM. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Since there was no indication that ferrous sulphate elicits an SI ≥ 3 when tested at 50%, it was considered not to be a skin sensitizer. No EC3 value (the estimated test substance concentration that will give a SI =3) could be established. Based on these results,

ferrous sulphate (and iron trinitrate) would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.


Migrated from Short description of key information:
A mouse LLNA is available for the read-across substance iron sulphate.

Justification for selection of skin sensitisation endpoint:
Only one study is available for this endpoint

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the negative result in a mouse LLNA performed with the read-across substance iron sulphate, no classification is proposed for iron trinitrate.