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Description of key information

One reliable animal study, performed according to OECD guideline 429 and according to GLP, shows that chromium iron oxide has no sensitisng properties.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation, other
Remarks:
in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-03-16 to 2010-04-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, 2002-04-24
Deviations:
yes
Remarks:
modified OECD 429 ,method according to Ehlings et al. 2005
Principles of method if other than guideline:
The test was performed in accordance with the method according to Ehling et al (2005): An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round, Toxicology 212 (2005) 60-68 and Ehling et al (2005): An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round, Toxicology 212 (2005) 69-79.

Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive
(these values were fixed empirically during the inter-laboratory validation of this method). In addition, the lymph node weights were determined for concentration related properties.
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-11-12
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 22- 27 g
- Housing: The animals were kept singly in MAKROLON cages (III) with a basal surface of approx. 39 cm X 23 cm and a height of approx. 15 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany was used as bedding material for the cages.
- Diet (ad libitum): Commercial diet ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C (maximum range)
- Relative humidity: 55% +/- 15 % (maximum range)
- Air changes: 12 -18 times per hour
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.
Vehicle:
other: acetone/olive oil (3 + 1, v/v)
Concentration:
10 % w/w, 25 % w/w, and 50 % w/w of Colorante negro
No. of animals per dose:
6 female mice
Details on study design:
RANGE FINDING TESTS:
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 10, 25 and 50 % of Colorante negro in acetone/olive oil (3 + 1 v/v), w/w, were examined.
Results:
No pronounced irritating properties were observed in this preliminary experiment at concentrations of 10 %, 25 % or 50 %. No differences in ear weight and ear thickness were noted.

MAIN STUDY
The test item was suspended in a mixture of acetone (Batch no. 90980, SIGMA ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany) / olive oil (Batch nos. 4535401, Henry Lamotte GmbH, 28197 Bremen, Germany) (3 + 1), v/v). The vehicle was selected on the basis of maximising the test concentrations and solubility whilst producing a solution/suspension suitable for application of the test item.

The test item suspension was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.

The concentrations used in the main study were chosen based on the preliminary dose range finding.

The experimental schedule of the assay was as follows:
Day 1:
The weight of each animal was individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Day 4 (24 hours after the last application the animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS/0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

OBSERVATIONS:
The following observations were made during the course of the study:

- Clinical signs: Animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Observations were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:30 a.m. to 4:30 p.m. On Saturdays and Sundays animals were checked regularly from 8:00 a.m. to 12:00 noon with a final check performed at approximately 4:00 p.m., if applicable.
- Body weight: The weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4).

ANALYSIS OF RESULTS:
The so-called stimulation (or LLN-) indices to determine the sensitising potential were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.
For lymph node weight significance at p≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. Outliers would have been determined according to the Nalimov test.
In addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.
The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.
No further information on the study design was stated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Please refer to "details on study design"
Positive control results:
The positive control group caused the expected increases in lymph node cell count (statistically significant at p≤ 0.01). The value for the stimulation index of lymph node cell count was 1.78. Even though also the lymph node weight was increased in the positive control (indicating factor to be a substance with irritating properties), the no observed "acute skin irritation" indicates that the positive control group has sensitising properties. Therefore, the study could be regarded as valid.
Ear weight: SI: 1.083
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method). 10 % w/w: SI: 1.013 (cell count); SI: 1.213 (lymph node weight) 25 % w/w: SI: 1.022 (cell count); SI: 1.404 (lymph node weight) 50 % w/w: SI: 1.292 (cell count); SI: 1.915 (lymph node weight)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable

Treatment with Colorante negro at concentrations of 10 %, 25 % or 50 % did not reveal statistical significantly increased values for lymph node cell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitising in the course of this study.

The threshold level for the ear weight of 1.1 was not exceeded; however, the lymph node weights were increased in a dose-related way. This finding could point to irritating properties of the test item specifically in mice although no acute skin reactions could be observed.

(Please refer to "Attached background material" for the raw data on stimulation indices)

No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions, Colorante negro at concentrations of 10 %, 25 % or 50 % (w/w) in acetone/olive oil (3 +1, v/v) did not reveal any sensitising properties in the local lymph node assay and therefore should not be classified and labelled according to regulation (EC) No.: 1272/2008.
Also, according to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is not classified as skin sensitiser.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The positive control group caused the expected increases in lymph node cell count (statistically significant at p≤ 0.01). The value for the stimulation index of lymph node cell count was 1.78. Even though also the lymph node weight was increased in the positive control (indicating factor to be a substance with irritating properties), the no observed "acute skin irritation" indicates that the positive control group has sensitising properties. Therefore, the study could be regarded as valid. Ear weight: SI: 1.083



Respiratory sensitisation

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

Migrated from Short description of key information:
Chromium iron oxide does not show respiratory sensitising properties in exposure related observations in humans.

Justification for classification or non-classification

Sensitisation:

Under the present test conditions, Colorante negro at concentrations of 10 %, 25 % or 50 % (w/w) in acetone/olive oil (3 +1, v/v) did not reveal any sensitising properties in the local lymph node assay and therefore should not be classified and labelled according to regulation (EC) No.: 1272/2008. Also, according to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is not classified as skin sensitiser.

Respiratory sensitisation

There is no evidence on specific respiratory hypersensitivity in humans following repeated inhalation exposure with chromium iron oxide. Thus, the classification criteria acc. to regulation (EC) 1272/2008 as respiratory sensitiser are not met.