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Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Batch No. is given, guideline 406 has been followed and study is performed according to GLP.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Principles of method if other than guideline:
In the report, it had not been clearly mentioned whether the epidermal application during induction was semi-occlusive or occlusive. Additional information from the test lab learnt that it was occlusive.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: approximately 5 weeks
- Weight at study initiation: 361 - 407 g
- Housing: 1 or 2 animals per cage with wire-mesh floors
- Diet (e.g. ad libitum): free access to standard guinea pig diet
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day


IN-LIFE DATES: From: February 5, 1996 To: February 29, 1996
Route:
other: intradermal and epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Induction: 5% (intradermal), 50% test material concentration (epicutaneous)
Challenge: 50%, 25% and 10% test material concentrations and the vehicle (distilled water)
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Induction: 5% (intradermal), 50% test material concentration (epicutaneous)
Challenge: 50%, 25% and 10% test material concentrations and the vehicle (distilled water)
No. of animals per dose:
- Main test:
Experimental group: 10 females
Control group: 5 females
- Preliminary test: 4 animals in total
Details on study design:
RANGE FINDING TESTS:
- By intradermal route tested concentrations: in 1 animal a 25% and a 10% concentration (0.1 mL each injected), in another animal a 5% and a 2% concentration
- By epidermal route:
1. after intradermal injection: in 1 animal 50% (0.5 mL), in the other animal 25% concentration
2. 50%, 25%, 10% and 5% (0.05 mL each) in two other animals

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures:
Intradermal injections (3 pairs and 0.1 ml/site):
1. FCA, 50:50 with water
2. test material at 5% concentration
3. test material in a 50:50 mixture of FCA
Topical application: After treatment with SDS a patch with 0.5 mL of 50% test material was applied
Controls: treated as experimental animals except that vehicle only was administered
- Exposure period: topical application 48 hours
- Test groups: test material
- Control group: vehicle (distilled water)
- Site: (clipped) scapular region
- Frequency of applications: 7 days after the intradermal injection the sites were treated with the test sample for 48 h
- Duration: from injection till topical application was 7 days
- Concentrations: 5% intradermal and 50% epidermal


B. CHALLENGE EXPOSURE
- No. of exposures:
Topical application: on day 22 all animals were challenged by a cutaneous application of the test material. 4 concentrations were applied and maintained for 24 hours.
- Day(s) of challenge: 24 hours (3 weeks after the first intradermal injection)
- Exposure period: 24 h
- Test groups: test material
- Control group: test material
- Site: a clipped flank
- Concentrations: 50%, 25%, 10% and the vehicle, as occlusive applications
- Evaluation (hr after challenge): evaluated approximately 24 and 48 hours after removal of the dressing
Grading system:
Score
No visible change 0
Discrete or patchy erythema 1
Moderate and confluent erythema 2
Moderate erythema and swelling 3
Intense erythema and swelling 4



OTHER:
Challenge controls:
Yes, treated with test material.
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamicaldehyde, tech. 85%
Positive control results:
- Test material:
ALPHA-HEXYLCINNAMICALDEHYDE, TECH. 85% (CAS no. 101-86-0) was fabricated under lot no. 80281 and the purity was 99.4% (GLC) (Aldrich Chemicals Co., Germany).
- Results: The skin reactions in the experimental animals observed in response to the 50% and 25% test substance concentrations in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals.
These results lead to a sensitisation rate of 100 per cent for the 50% concentration and to a sensitisation rate of 60 per cent for the 25% concentration.
From these results, it was concluded that the Albino Dunkin Hartley strain of guinea pig is an appropriate animal model for the performance of studies designed to evaluate the sensitising potential of a substance in a Maximisation type of test.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
24 h respectively 48 h after challenge, the test and control animals did not show specific primary skin irritations or a reference to a specific sensitive effect. The test material FeEDDHMANa should not be classified as sensitizing to the skin.
Executive summary:

A Guinea Pig Maximisation test was conducted with a group of 10 Dunkin Hartley guinea pigs and a negative control group of 5 Dunkin Hartley guinea pigs. They were used to investigate the skin sensitising properties of the FeEDDHMANa (trade name Bolikel FE). The study was performed in accordance with the method of B. Magnusson and A. M Kligman (OECD-guidelines: Guideline 406 ‘Skin Sensitisation’). The study was designed and performed according to Good Laboratory Practice Standards.

The test group received first intradermal injections with 5% test substance. After 7 days a topical application of 10% SDS in vaseline was performed. On Day 8 the animals were treated with a 48 h occlusive epicutaneous application of 50% concentration in distilled water. Two weeks after the last application, the test animals received a 24 h occlusive epicutaneous application (concentrations 50%, 25%, 10% and 0%). After 24 h and 48 h skin reactions were scored. Controls were treated with vehicle (distilled water) during induction and similar as the treated group during challenge.

No clinical signs and no deaths were noted during the study.

After the induction phase some skin effects were seen, like brown staining of the skin, small scabs, erythema and necrosis. The reactions were considered to be enhanced by SDS treatment.

After the challenge application, a discrete erythema was recorded in 1/10 animals of the 25% treated group at the 24- and 48-hour reading. The response to the 25% concentration was considered non-specific, since no response was observed to the higher concentration (50%) in this animal. No skin reactions were evident in the control animals.

24 h respectively 48 h after challenge, the test and the control animals did not show specific primary skin irritations or a reference to a specific sensitive effect. The test material FeEDDHMANa should not be classified as sensitizing to the skin.

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

Six out of eight guinea pig maximisation tests showed no skin sensitising properties of EDDHMA-Fe. All these tests (except for one, Kynoch (1977), pre-GLP) were carried out according to OECD 406 and according to GLP. Two older tests, however, showed skin sensitising properties. One test was carried out in 1990 (Smith), also carried out according to GLP and OECD 406; the other test (Skydsgaard, 1987) was not carried out according to GLP and was a modification of the Magnusson and Kligman test using four topical applications.

The fact that more recent studies using higher induction and challenge concentrations did not show sensitising properties whereas two older studies did, allows the conclusion that the older products may have contained one (or more) impurity (/impurities) that had sensitising properties. Overall it is, therefore, concluded that EDDHMA-Fe is not a skin sensitiser. In addition it is noted that EDDHMA-Fe does not interact with protein, which is considered a requirement for sensitization (needed for haptenisation). Finally, also decades of use of this substance did not result in any reports of people becoming sensitized.


Migrated from Short description of key information:
Several tests were available in which skin sensitization has been studied. Six studies showed no skin sensitization properties whereas two studies showed slight skin sensitization properties.

Justification for selection of skin sensitisation endpoint:
One key study with 5 supporting studies showed no skin sensitizing properties

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:
Migrated from Short description of key information:
Decades of use of this substance did not result in any reports of people becoming sensitized.

Justification for classification or non-classification

Six available studies (OECD 406 and GLP) did not indicate sensitising properties of EDDHMA-Fe. Two older studies indicate either a possible weak, or an ambiguous result for a weak potential for sensitisation of EDDHMA-Fe. The substance does not interact with protein and years of use of this substance did not result in any reports of people becoming sensitized. In conclusion, in the evaluation of all available information, EDDHMA-Fe should not be classified with regard to sensitisation according to Directive 67/548/EEC (DSD) and to Regulation (EC) No 1272/2008 (CLP), respectively.