Chromium (III) complexes with 2-[{2,4-dihydroxy-3-[(5-sulfo-1-naphthyl)diazenyl]phenyl}diazenyl]benzoic acid (1:2), sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 2015-28 March 2015

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Chromium (III) complexes with 2-[{2,4-dihydroxy-3-[(5-sulfo-1-naphthyl)diazenyl]phenyl}diazenyl]benzoic acid (1:2), sodium salts

EC Number:

700-953-9

Molecular formula:

C46H26CrN8O14S2.3Na

IUPAC Name:

Chromium (III) complexes with 2-[{2,4-dihydroxy-3-[(5-sulfo-1-naphthyl)diazenyl]phenyl}diazenyl]benzoic acid (1:2), sodium salts

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test system justification:
The mouse is the recommended species for this test method according to international test guidelines. The BALB/c strain is selected because of the availability of historical control data for this strain at this facility, and because it was regarded as a sensitive strain in the context of contact allergy.

Experimental animals:
Animals species and strain: CBA/Ca Ola Hsd mice
Source: TOXI-COOP ZRT.-H-1103, Budapest, Cserkesz u. 90.
Number of animals: 24 animals/main test (4 animals/treatment group)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice; 9-10 weeks old (at start of the main test)
Body weight range: at starting: 18.5-21.6 g
The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
Acclimatization time: 21 days

Husbandry
Animal health: Only healthy animals were used
Housing during
acclimatization period: Grouped caging in small groups
Housing during the test: G Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
Housing/Enrichment: Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
There were no deviations from these specifications during the experimental phase. The temperature and relative humidity were recorded during the acclimatization and experimental phases. Before housing the animals, the microbiological status of the room was checked

Study design: in vivo (LLNA)

Vehicle:

other: 1 % Pluronic

Concentration:

1-2,5-5-10 %(w/v)

No. of animals per dose:

Positive control group – 4 females
Vehicle of the positive control: 4 females
Test item: 4 concentrations -4 females each
Vehicle of the test item: 4 females
Total: 28 animals

Details on study design:

The pre-experiments on solubility of the test item and the Dose Range Finding Test was not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study code of present study.
The test item was formulated in aqueous 1 % Pluronic and evaluated at concentrations of 10 % and 5 % (w/v). Both formulations (apparently solutions) were adequately applicable on the ears of animals.
Two groups of 2 CBA/Ca mice were treated with the appropriate formulations once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the preliminary test.
Body weights were recorded prior to the first treatment (on Day 1) and prior to termination (on Day 6). Both ears of each mouse were observed for erythema and scored according to criteria depicted in Table 2. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
No mortality was observed. Although body weight loss (5 %) was observed in both dose groups (1/2 animals in each) it was considered not treatment related, since no body weight loss was observed for the other animals in the dose groups. Additionally, no any other sign of systemic toxicity were observed during the preliminary test. No signs of significant irritation (indicated by an erythema score ≥ 3 and/or an increased ear thickness of ≥ 25 % on any day of measurement) were observed in the treatment groups. Tabulated results of the above preliminary test are given in Tables 4-7 of Appendix I.
Based on the preliminary test results the maximum attainable concentration (based on solubility in an appropriate vehicle) of 10 % (w/v) was used in the main test. The test item was tested also at three additional, lower concentrations (5 %, 2.5 % and 1 %, w/v) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.
Main Test Design
Animals in the treatment groups were treated with the relevant vehicles (aqueous 1 % Pluronic or AOO), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the dose range finding test.
In vivo Treatment
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control item or the negative controls (aqueous 1 % Pluronic or AOO) using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
Proliferation Assay
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technically failed treatment was observed during the test: all animals treated were processed. Therefore no any treatment group was excluded from the evaluation.

Positive control substance(s):

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

Statistics:

The test item is considered as a skin sensitizer, if:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

Results and discussion

Positive control results:

Clinical Observations
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.
Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.
Evaluation of the Results
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value.
The results were expressed as DPN (DPM divided by the number of pooled lymph nodes). The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
Significance of the dose-response relationship was evaluated by linear regression using the obtained SI values. All calculations were made by Microsoft Excel Software. Based on the results EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

S.I.V.: Vehicle positive control: 1.0 Positive control: 5.7 Test item 10% 1.3 Test item 5% 1.4 Test item 2,5% 1.1 Test item 1% 1.0 Test item vehicle 1.0

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

DPM Group: Vehicle positive control: 5514 Positive control: 31269 Test item 10% 2950 Test item 5% 3028 Test item 2,5% 2515 Test item 1% 2139 Test item vehicle 2188

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present assay, Acid Brown 432 tested at the maximum feasible concentration of 10% (w/v, based on solubility) and at concentrations of 5 %, 2.5 % and 1 % (w/v) as formulations in a suitable vehicle (aqueous 1 % (w/v) Pluronic®PE 9200) was shown to have no skin sensitization potential in the Local Lymph Node Assay.

Executive summary:

The maximum dose selection was performed according to the relevant guidelines [1-2] and based on results of a formulation evaluation and also results of the preliminary irritation/toxicity test. Based on the formulation evaluation the maximum attainable concentration (based on solubility) was 10 % (w/v) in aqueous 1 % (w/v) Pluronic®PE 9200 (aqueous 1 % Pluronic). Based on results of the preliminary irritation/toxicity test Acid Brown 432 was tested in the LLNA at concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v) as formulations in the selected vehicle of aqueous 1 % Pluronic. The test was valid and no confounding effect of irritation or obvious signs of systemic toxicity were considered to interfere with the results. The effect on the body weights observed was considered not significant or treatment related. Hence the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant vehicle control (aqueous 1 % Pluronic) was noted for Acid Brown 432 up to the maximum feasible concentration of 10 % (w/v). No significant dose-response relationship was observed.

According to evaluation criteria of the relevant guidelines [1,2], since no SI ≥ 3 was observed up to the maximum feasible concentration of 10 % (w/v, based on solubility in an appropriate vehicle) and the lack of a significant dose-related response are considered evidence that Acid Brown 432 is not a skin sensitizer.