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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:
06 Sep - 05 Oct 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 23 Jul 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Health Care Inspectorate of the Ministry of Health, Welfare and Sport, Utrecht, The Netherlands
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Hexanedioic acid, di-C16-18-alkyl esters
EC Number:
296-703-7
EC Name:
Hexanedioic acid, di-C16-18-alkyl esters
Cas Number:
92969-90-9
Molecular formula:
not applicable, substance is UVCB
IUPAC Name:
Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Remarks:
SPF quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: 17.5 - 21.5 g (Group 1); 20.8 - 22.7 g (Group 2); 19.5 - 23.3 g (Group 3); 22.0 - 26.3 g (Group 4)
- Housing: 5 females per group were housed in labeled Makrolon Cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material. Paper and shelters were supplied as cage-enrichment.
- Diet: SM R/M-Z, pelleted rodent diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: Animals were healthy and the ears were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25 and 50% (w/w)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS: 2 female mice per concentration were treated by daily application of a 50% in dimethylformamide or undiluted test substance to the dorsal surface of the ear, for 3 consecutive days. Very slight erythema (Grade 1) and/or scaliness were noted for the animals treated with the undiluted test substance between Days 2 - 6. Hunched posture and/or piloerection were observed for the animals treated with the undiluted test substance on Days 2 and 3. With a 50% test substance concentration no signs of systemic toxicity were noted and no erythema were observed, but scaliness was noted for one animal between Days 4 and 6.
- Irritation: The animals were observed for local skin irritation to the application site once daily on Day 1 (pre-dose) to Day 6 (prior to necropsy). On Days 1-3 observation was performed within 1 h after dosing.
- Systemic toxicity: The animals were observed for mortality twice daily. The body weight was recorded on Day 1 prior to dosing and on Day 6. Signs of toxicity were recorded on Days 1-6 (on Days 1-3 between 3-4 h after dosing).
- Ear thickness measurements: Ear thickness measurements of both ears were performed using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.
- Erythema scores: Draize scoring system

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine incorporation determined by β-scintillation and γ-counting
- 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 using the individual SI values. The individual SI is the ratio of the DPM/animal compared with the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item is regarded as a skin sensitizer.
- Other: The animals were observed for mortality twice daily. The body weight was recorded on Day 1 prior to dosing and on Day 6. Signs of toxicity were recorded on Days 1-6 (on Days 1-3 between 3-4 h after dosing).

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of the test material was applied to the entire dorsal surface of each ear of each mouse on Day 1, 2 and 3 in concentrations of 10, 25 and 50% in dimethylformamide. The local irritation effects on the treatment site were assessed daily. On Day 6 an injection of 250 µL phosphate buffered saline (PBS) containing 20 µCi of ³H-methyl thymidine (³H-TdR) was made into the tail vein of each mouse. Five hours later, the draining auricular lymph node of each ear was excised into PBS and pooled per group. A single cell suspension of pooled lymph node cells was prepared by gentle separation through a 200-mesh stainless steel gauze and rinsed with PBS. The precipitates were incubated in the refrigerator until the next day, centrifuged, resuspended in 1 mL trichloroacetic acid and transferred to 10 mL scintillation fluid before β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed at half-yearly intervals during at least the past 9 years, showing reproducible and consistent positive results with hexyl cinnamic aldehyde. The SI values calculated for the positive control concentrations 5, 10 and 25% were 1.4, 1.5 and 4.3, respectively. An EC3 value of 18.0% was calculated using linear interpolation (Test facility study number: 513924, May 2016).
Based on the results, the positive control was considered to demonstrate the appropriate performance of the assay, with adequate and reproducible sensitivity to a known sensitising test substance.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
10% (w/w)
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
25% (w/w)
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
50% (w/w)
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION: The SI of the 10, 25 and 50% treatment group was 0.9, 1.1 and 1.4, respectively. None of the test substance concentrations produced as 3-fold increase in ³HTdR incorporation.

EC3 CALCULATION: None of the SI values were above 3 and it is therefore not possible to determine a EC3 concentration.

CLINICAL OBSERVATIONS: No mortality occured and no signs of systemic toxicity were observed in any of the animals. Very slight erythema (Grade 1) was observed in 3/5 females treated with a 50% test substance concentration (w/w) on Day 2, 3 and 4, respectively.

BODY WEIGHTS: The body weights and body weight gain remained with the same range as controls during the study period.

Any other information on results incl. tables

Table 1: Irritation scores and clinical signs of toxicity recorded in the pre-screen test

Test substance concentration Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
left right left right left right left right left right left right
50% 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 S 0 0 S 0 0 S 0
100% 0 0 0 H 1 1 HP 1 0 S 0 0 S 0 0 S 0 S
0 0 0 HP 0 1 HP 1 0 S 0 S 0 S 0 S 0 S 0 S

H: Hunched posture

P: Piloerection

S: Scaliness

0: no erythema; 1: very slight erythema

Table 2: Results of the main test

Test substance DPM/animal mean DPM± SEM mean SI ± SEM
Solvent control 1256 748± 205 1.0± 0.4
74
537
1020
851
10% 668 704± 48 0.9± 0.3
809
573
647
822
25% 845 790 ± 57 1.1± 0.3
905
88
619
694
50% 871 1070 ± 225 1.4± 0.5
879
490
1809
1302

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008
Conclusions:
CLP: not classified