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Description of key information

FAT 93460 was considered to be a non-sensitizer in a mouse local lymph node assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 July 2016 to 23 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
EC 440/2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese MAFF
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identification: FAT 93460/D TE
Batch: 20140721 (China)
Physical state/Appearance: Yellow powder
Purity: 94.6 %
Expiry Date: 14 October 2019
Storage Conditions: approximately 4 °C in the dark.
Species:
mouse
Strain:
CBA/Ca
Remarks:
(CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
Animal Information
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

Animal Care and Husbandry
The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
dimethylformamide
Concentration:
Preliminary Screening test: 25 % w/v
Main test: 5 %, 10 % and 25 % w/v
No. of animals per dose:
Preliminary screening test = 1
Main test = 4 per dose
Details on study design:
Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at a maximum attainable concentration of 25 % w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Annex 4. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25 % was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 25 %, 10 % or 5 % w/w in dimethyl formamide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5 % Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by -scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
Positive control results:
A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitizer. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals No. 429 and Method B.42 of Commission Regulation (EC) No. 440/2008.
Test item: α-Hexylcinnamaldehyde, tech., 85 %
Study number: 41502435
Study dates: 04 November 2015 to 10 November 2015
Methods: A group of five animals was treated with 50 μL (25 μL per ear) of α-Hexylcinnamaldehyde, tech., 85 % as a solution in dimethyl formamide at a concentration of 25 % v/v. A further control group of five animals was treated with dimethyl formamide alone.

Results; The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in dimethyl formamide Stimulation Index Result
25 6.08 Positive
Conclusion: α-Hexylcinnamaldehyde, tech., 85 % was considered to be a sensitizer under the conditions of the test.
Key result
Parameter:
SI
Value:
0.96
Test group / Remarks:
5% w/v
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
10% w/v
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
0.87
Test group / Remarks:
25% w/v
Remarks on result:
other: Negative
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS: Yellow colored staining on the ears was noted in all test animals. Yellow-colored residual test item on the ears was also noted in animals treated with the test item at a concentration of 25 % w/w in dimethyl formamide. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitizer under the conditions of the test.
Executive summary:

A study was performed according to OECD test guideline 429 in a GLP certified laboratory to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods: Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable concentration of 25 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as an emulsion in dimethyl formamide at concentrations of 25 %, 10 % or 5 % w/w. A further group of four animals was treated with dimethyl formamide alone.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in dimethyl formamide       Stimulation Index              Result

5                                                          0.96                            Negative

10                                                        1.10                            Negative

25                                                        0.87                            Negative

Conclusion:

The test item was considered to be a non-sensitizer under the conditions of the test. The test item does not meet the criteria for classification according to the Globally Harmonized Classification System.

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

The skin sensitization potential of FAT 93460 was evaluated in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable concentration of 25 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as an emulsion in dimethyl formamide at concentrations of 25 %, 10 % or 5 % w/w. A further group of four animals was treated with dimethyl formamide alone. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were determined as 0.96, 1.10 and 0.87 at 5, 10 and 25% w/w concentrations respectively, giving a negative result. Hence, FAT 93460 was considered to be a non-sensitizer under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the outcome of a local lymph node assay, FAT 93460 does not warrant classification under CLP (Regulation 1272/2008) criteria.