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Skin sensitisation

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Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 March 2012 - 16 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
see method; sufficient information available for evaluation.
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)
Remarks:
issued 19 May 2011
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeled makrolon cages
- 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): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Temporary deviation from the minimum level of daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 0.1, 0.25, 0.5%
No. of animals per dose:
5
Details on study design:
The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
Initially, two test substance concentrations were tested; a 50% and 100% concentration. 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. On day 3 mice were killed because of severe local effects at the treated sites (severe red ears and high ear thickness (>25%) on Day 3), lack of body weight gain and (at 100%) hunched posture on Day 3. Based on the results of the initially treated animals, six additional animals were treated in a similar manner with three lower concentrations (1%, 2.5% and 5%) at a later stage. No erythema, or very slight erythema (at 2.5 aand 5%) was observed in this group. Substantial percentual increase of ear thickness was meassured in all three groups, at day 6 (average of both ears of 2 animals): 25.25%, 53.25%, 98.25% for 1, 2.5 and 5% test substance respectively.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

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.

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.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: All animals surviving to the end of the study were sacrificed by intra-peritoneal injection with Euthasol® 20% (0.2 mL/animal). No necropsy was conducted on the animals of the pre-screen test.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.
Positive control results:
The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 0.25 and 0.5% were 3.6 and 6.2 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 0.25 and 0.5% were 2075 and 3584 DPM respectively. The mean DPM/animal value for the vehicle control group was 578 DPM.

Results Pre-screen test:

No erythema and no signs of systemic toxicity were observed in any of the animals at the lowest concentration of 1%. At this concentration the variations in ear thickness during the observation period were still slightly above 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 0.5% concentration.

Other results - main study:

No irritation of the ears was observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values

 

All auricular lymph nodes of the animals of the experimental were considered larger in size compared to the control groups No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, CX-100 was considered to be a skin sensitiser, as the SI appeared to be ≥ 3 when tested up to 0.5%.
Executive summary:

An LLNA skin sensitisation study, performed according to OECD/EC test guidelines, was performed with CX-100. Test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, two experimental groups of five female CBA/J mice were treated with test substance concentrations of 0.25 or 0.5% w/w on three consecutive days, by open application on the ears. Low dose animals were dosed at 1.68% instead of 0.1%, due to incorrect test substance weighing. The data from this group were not used for interpretation of the results. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). No irritation of the ears was observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. No mortality occurred and no clinical signs of systemic toxicity were observed. The SI values calculated for the substance concentrations 0.25 and 0.5% were 3.6 and 6.2 respectively. Since the test substance elicits an SI ≥ 3, Crosslinker CX-100 should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

An LLNA skin sensitisation study, performed according to OECD/EC test guidelines, was performed with the substance, Polyfunctional polyaziridine, based on TMPTA and Propylene imine. Test substance concentrations selected for the main study were based on the

results of a pre-screen test. In the main study, two experimental groups of five female CBA/J mice were treated with test substance concentrations of 0.25 or 0.5% w/w on three consecutive days, by open application on the ears. Low dose animals were dosed at 1.68% instead of 0.1%, due to incorrect test substance weighing. The data from this group were not used for interpretation of the results. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). No irritation of the ears was observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. No mortality occurred and no clinical signs of systemic toxicity were observed. The SI values calculated for the substance concentrations 0.25 and 0.5% were 3.6 and 6.2 respectively.

Migrated from Short description of key information:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, the substance was considered to be a skin sensitiser, as the SI appeared to be ≥ 3 when tested up to 0.5%.

Justification for selection of skin sensitisation endpoint:
Only one study available performed according to OECD guidelines and GLP principles with a reliability score of 1.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Since the substance elicits at a concentration of 0.25% an SI ≥ 3 in an LLNA test, this substance is classified according to CLP Regulation (EC) No. 1272/2008 as skin sensitizer (Category 1A) and labeled as H317 "May cause an allergic skin reaction".