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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 25 - June 29, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
impurity 1
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: 2016JAN-15kg
Expiry Date: 01 January 2020
Appearance: Blue-white powder
Storage: Room temperature (15-25°C)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Specific details on test material used for the study:
Batch No.: 2016JAN-15kg
Expiry Date: 01 January 2020
Appearance: Blue-white powder
Storage: Room temperature (15-25°C)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/Ca Ola Hsd mice
Source: TOXI-COOP ZRT.
Hygienic level at arrival: SPF
Hygienic level during
the test: Good conventional
Number of animals: 24 animals/main test (4 animals/treatment group)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice; 8-11 weeks old (at start of the main test)
Body weight range
at starting: 17.5 – 21.4 g
Acclimatization time: 28 or 7 days
Housing during the test: 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 %
Water and food consumption: ad libitum
In-life phase: June 01-07, 2016


Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
100, 50 and 25 %, w/v
No. of animals per dose:
4 animals/treatment group
Details on study design:
Based on results of the a Dose Range Finding test the test item was examined in the main test at concentrations of 100 %, 50 % and 25 % (w/v) as suspensions prepared with DMF. Three groups of 2 CBA/Ca mice were treated with the appropriate formulations. All formulations were adequately applicable on the ears of animals. 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:

Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

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.

Main Study:
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.

Treatment, preparation and administration:
Animals in the treatment groups were treated with the negative (vehicle) controls (DMF or AOO), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at three different concentrations according to the results of the dose range finding test.

Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. Test item formulations were applied on the ears by using end-cut pipette tips. 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. On Day 6 each mouse was intravenously injected via the tail vein with 250 microL of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 μCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).

Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation.

Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 Celsius. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse 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. Dose-response relationship was evaluated by linear regression using SI values. All calculations were made by Microsoft Excel Software. Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. Significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 7.7). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

In vitro / in chemico

Other effects / acceptance of results:
Significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 7.7). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

In vivo (LLNA)

Results
Key result
Parameter:
SI
Value:
> 1 - < 2.2
Variability:
SI of 100% concentration: 1.3; SI of 50% concentration: 1.0; SI of 25% concentration: 2.2
Test group / Remarks:
Test item was examined in the main test as 100 %, 50 % or 25 % (w/v) formulations (suspensions) in DMF.
Cellular proliferation data / Observations:
Since no failed treatment, sign of systemic toxicity or irritation was observed during the test no treatment group was excluded from the evaluation. Visually larger lymph nodes compared to the vehicle control (AOO) were observed in the positive control group only. Appearance of the lymph nodes was normal in the negative control groups (AOO or DMF) and in the test item treated groups.

No significant (SI ≥ 3) lymphoproliferative response compared to the relevant control (DMF) was noted for Neodymium fluoride oxide, magnesium doped at the applied test concentrations. The observed stimulation index values were 1.3, 1.0 and 2.2 at test item concentrations of 100 %, 50 % and 25 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.61, r = 0.58).

The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse 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. Dose-response relationship was evaluated by linear regression using SI values. Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group. No significant, treatment related effect on body weights was observed during the test. Although body weights, decreased by ≥ 5 % were observed in the 50 % and 25 % (w/v) dose groups (1/4 animals in each, 8 % or 5 % decrease, respectively) the effect was considered neither significant nor treatment related.


Any other information on results incl. tables

The pre-experiments on formulation evaluation and the Dose Range Finding Test (DRF) were not performed in compliance with the GLP-Regulations and are excluded from the Statement of the Study Director, but the raw data of these tests are archived under the study code of present study. The preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main test except there was no assessment of lymph node proliferation and fewer animals were used. The maximum test concentration was selected according to results of the preliminary formulation evaluation. The test item was examined in the DRF as 100 % (i.e. 1 g/mL), 50 % or 25 % (w/v) formulations (suspensions) prepared with the selected vehicle of DMF. Three groups of 2 CBA/Ca mice were treated with the appropriate formulations. All formulations were adequately applicable on the ears of animals. 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 the study criteria. 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, significant, treatment related effect on the body weights or any other sign of systemic toxicity were observed. No sign of significant irritation (indicated by an erythema score  3 and/or an increase of  25 % of ear thickness observed on any day of measurement) or any other local effect were observed.

Based on the results 100 % (w/v) test item concentration was selected to be used as the maximum in the main test.

The test item was tested also at two additional, lower concentrations (50 % and 25 %, w/v) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.

Applicant's summary and conclusion

Interpretation of results:
other: No skin sensitisation potential
Conclusions:
Under the conditions of the present assay, Neodymium fluoride oxide, magnesium doped tested at the maximum feasible concentration of 100 % (1 g/mL) and at concentrations of 50 % and 25 % (w/v) as formulations (apparently suspensions) in a suitable vehicle (DMF) was shown to have no skin sensitization potential in the Local Lymph Node Assay.
Executive summary:

Since no failed treatment, sign of systemic toxicity or irritation was observed during the test no treatment group was excluded from the evaluation. No significant (SI  3) lymphoproliferative response compared to the relevant control (DMF) was noted for Neodymium fluoride oxide, magnesium dopedat the applied test concentrations. The observed stimulation index values were 1.3, 1.0 and 2.2 at test item concentrations of 100 %, 50 % and 25 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.61, r = 0.58).

The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse 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. Dose-response relationship was evaluated by linear regression using SI values. All calculations were made by Microsoft Excel Software.Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation or any other local effect were observed in any treatment group. No significant, treatment related effect on body weights was observed during the test. Although body weights, decreased by ≥ 5 % were observed in the 50 % and 25 % (w/v) dose groups (1/4 animals in each, 8 % or 5 % decrease, respectively) the effect was considered neither significant nor treatment related.

Under the conditions of the present assay, Neodymium fluoride oxide, magnesium doped tested at the maximum feasible concentration of 100 % (1 g/mL) and at concentrations of 50 % and 25 % (w/v) as formulations (apparently suspensions) in a suitable vehicle (DMF) was shown to have no skin sensitization potential in the Local Lymph Node Assay.