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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:
March-May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD guideline and GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene
EC Number:
615-240-7
Cas Number:
710292-85-6
Molecular formula:
Not available for this UVCB
IUPAC Name:
Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene
Details on test material:
- Name of test material (as cited in study report): HK-128
- Physical state: red-violet waxy solid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: young adult animals (approx. 10 weeks old)
- Weight at study initiation: +/- 20% of the sex mean
- Housing: group housed in labeled Makrolon cages containing sterilised sawdust as bedding material; paper and shelters were supplied as cage-enrichment
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.1-22.0
- Humidity (%): 23-74
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Deviations from the minimum level of temperature and relative humidity occurred. Laboratory historical data do not indicate an effect of these deviations.

IN-LIFE DATES: 21 March - 16 April 2012

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 10, 25%; The highest concentration was the maximum that could be prepared homogeneously.
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor
- Irritation: A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation at the most (maximum grade 2 and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.
Two test substance concentrations were tested; a 10% and 25% concentration. The highest concentration was the maximum that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.
- Lymph node proliferation response: not determined in pre-screen test

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five
animals was treated with vehicle and another group of five animals was treated with the positive control
substance.

TREATMENT PREPARATION AND ADMINISTRATION
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance concentration, at approximately the
same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control
animals were treated the same as the experimental animals, except that the vehicle was administered instead of
the test substance.

Excision of the nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany)
containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five
hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).
The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was
estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled
for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6:
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze
(diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To
precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) stored in the
refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold
cocktail (Perkin Elmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements
were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a
maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert
Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On Days 1 (pre-treatment) and 6.
Clinical signs: once daily on days 1-6
Irritation: once daily on days 1-6 (on days 1-3 within 1 hour after dosing)
according to the following numerical scoring system; descriptions of all other (local) effects were
recorded.

Grading Irritation Reactions:

Erythema and eschar formation:
0: No erythema
1: Very slight erythema (barely perceptible)
2: Well-defined erythema
3: Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth)
4: Severe erythema (beet redness) to eschar formation preventing grading of erythema
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.

Results and discussion

Positive control results:
The EC3 value of 12.8% was in the acceptable range of 4.8-19.5% confirming the validity of the current test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
5% concentration
Value:
ca. 1.1
Test group / Remarks:
5 animals
Key result
Parameter:
SI
Remarks:
10% concentration
Value:
ca. 2.3
Test group / Remarks:
5 animals
Key result
Parameter:
SI
Remarks:
25% concentration
Value:
ca. 4.2
Test group / Remarks:
5 animals
Key result
Parameter:
EC3
Value:
ca. 15.5

Any other information on results incl. tables

Results pre-screen:

Very slight erythema was noted in animals at 25% on Days 2 and 3. Red test substance remnants were present on the dorsal surface of the ears of both animals at 10 and 25% (Days 1-3 and/or 4), which did not hamper scoring of the skin reactions. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

Other results main study:

Very slight erythema was noted in all animals at 25% on Days 2 and 3. This slight irritation of the ears was considered not to have a toxicologically significant effect on the activity of the nodes.

Red test substance remnants were present on the dorsal surface of the ears of all animals at 5, 10 and 25% (Days 1-3 and/or 4), which did not hamper scoring of the skin reactions. All auricular lymph nodes of the animals at 25% were considered larger in size compared to vehicle control. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The substance is a skin sensitiser (1B).
Based on the results:
- according to the recommendations made in the test guidelines, HK 128 would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), HK 128 should be classified as skin sensitizer (Category 1).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, HK 128 should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.