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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
QUALITY ASSURANCE ATTESTATION, G.L.P. COMPLIANCE STATEMENT & GLP certificates included in the full report. The study was conducted according to an internationally recognised method, and under GLP. No deviation was reported. The substance is adequately characterised. Therefore full validation applies.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Test method B.6 directive 96/54/EC
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
6,8-dimethylnon-7-enal
EC Number:
486-670-9
Cas Number:
899810-84-5
Molecular formula:
C11H20O
IUPAC Name:
6,8-dimethylnon-7-enal
Test material form:
other: liquid
Details on test material:
• Form : liquid
• Colour : colorless
• Batch n° : 862036
• Storage : room temperature
• Production date: September 2007 • Expiry date: September 2008
• Purity: 97.79%

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
For the main study, 15 female albino guinea pigs of Dunkin-Hartley strain, supplied by Charles River
(F-69592 L’Arbresle) weighed between 294 and 346 at the beginning of the test and were 5 weeks old.
Prior to the test, the animals were kept for a minimum acclimatisation period of 5 days, under stabling
and nutritional conditions identical to those of the test.
The animals were housed either in groups of 2 or 3 in polycarbonate containers, the flooring of which
was covered with dust-free cuttings and the top fitted a stainless steel lid with a feeding device and
drinking device of 500 ml.
The environmental parameters of the main test were :
- Temperature : between 19°C and 24°C
- Relative humidity : between 45% and 70%
- Lighting time: 12 hours daily
- rate of air exchange: at least ten changes per hour

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Remarks:
Topical application = paraffin
Concentration / amount:
Preliminary study;
Intradermal injection: 6 concentrations: 100% diluted at 50%, 25%, 12.5%, 6.25% and 3.125% in olive oil.
Topical application: 4 different concentrations: 100% diluted at 50%, 25% and 12.5% in liquid paraffin.

1st Intradermal Induction: test item at 6.25%.
2nd Topical Induction : test item at 100%
Challenge phase : test item at 12.5% (MNIC) and at 6.25% (1/2 MNIC).
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Remarks:
Topical application = paraffin
Concentration / amount:
Preliminary study;
Intradermal injection: 6 concentrations: 100% diluted at 50%, 25%, 12.5%, 6.25% and 3.125% in olive oil.
Topical application: 4 different concentrations: 100% diluted at 50%, 25% and 12.5% in liquid paraffin.

1st Intradermal Induction: test item at 6.25%.
2nd Topical Induction : test item at 100%
Challenge phase : test item at 12.5% (MNIC) and at 6.25% (1/2 MNIC).
No. of animals per dose:
in a first step 5 animals were retained for the control group and 10 for the treated one.
Details on study design:
Prior to the test, the animals were kept for a minimum acclimatisation period of 5 days, under stabling
and nutritional conditions identical to those of the test.
Before the experimentation process, they were identified individually by marking with picric acid and
a tattoo placed on their ear.
The animals were carefully shorn before each test item application:
- on the inter-scapular zone for the induction phase,
- on the dorso-lumbar zone for the challenge phase.
At least 3 hours before the first reading (challenge phase) they were shorn a second time in this dorsolumbar
zone.
The animals were weighed at the beginning and at the end of the study.
Positive control substance(s):
yes
Remarks:
α-Hexylcinnamaldehyde

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The concentrations of the test and reference items are indicated in the following table:
4 females Vehicle 0 (undiluted)
4 females test item 25%
4 females test item 50
4 females test item 100
No. of animals per dose:
4
Details on study design:
Preliminary study
As toxicological information was available regarding the systemic toxicity/irritancy potential of the
test item, a preliminary screening test was not performed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
2.5 - Interpretation of results
The proliferation response of lymph node cells was expressed as the number of lymphocytes per
lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that
recorded for the control nodes (Stimulation Index).
Results are expressed as the Stimulation Index (SI).
When using the pooled approach, the SI is calculated according to the following formula:
cell count of treated group
SI =
cell count of control group
The test item will be regarded as a sensitiser if at least one concentration of the test item results is
greater than 1.4 compared to control values.
Other relevant criteria such as dose-response and irritation level were also taken into account for the
interpretation of the results.
Any test item failing to produce a SI < 1.4 will be classified as a "non-sensitiser".
2.6 Determination of the EC1.4 value
The EC1.4 value (theoretical concentration resulting in a SI value of 1.4) was determined by linear
interpolation of points on the dose-response curve, immediately above and below the 1.4-fold
threshold. The equation used for calculation of EC1.4 was:
EC1.4 = c + [(1.4 – d) / (b – d)] x (a – c)
Legend: a = the lowest concentration giving stimulation index > 1.4
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.4
d = the actual stimulation index caused by c

Results and discussion

Positive control results:
Please see Appendix 2 Full report.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
6 %
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 6 %. No with. + reactions: 10.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
12 %
No. with + reactions:
50
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 12 %. No with. + reactions: 50.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
6 %
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 6 %. No with. + reactions: 10.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
12 %
No. with + reactions:
30
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 12 %. No with. + reactions: 30.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
6 %
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: 6 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
12 %
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: 12 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
6 %
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: 6 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
12 %
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: 12 %. No with. + reactions: 0.0. Total no. in groups: 5.0.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Interpretation of results The proliferation response of lymph node cells was expressed as the number of lymphocytes per lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). Results are expressed as the Stimulation Index (SI). When using the pooled approach, the SI is calculated according to the following formula: cell count of treated group SI = cell count of control group The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values. Other relevant criteria such as dose-response and irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI < 1.4 will be classified as a "non-sensitiser".
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Determination of the EC1.4 value The EC1.4 value (theoretical concentration resulting in a SI value of 1.4) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.4-fold threshold. The equation used for calculation of EC1.4 was: EC1.4 = c + [(1.4 – d) / (b – d)] x (a – c) Legend: a = the lowest concentration giving stimulation index > 1.4 b = the actual stimulation index caused by a c = the highest concentration failing to produce a stimulation index of 1.4 d = the actual stimulation index caused by c

Any other information on results incl. tables

RESULTS

1 - Concentrations selected

Preliminary studies:

- MNNC determination:

No necrosis has been observed, since the concentration of 6.25%. The first induction of the Group 2 has been carried out by intradermal injection at the same concentration of 6.25%.

-Pre MNIC determination:

24 hours after the removal of the occlusive dressings, no cutaneous reaction was recorded whatever the tested concentration.

In view of these results, the concentration selected was 100% for the 2nd induction of the Group 2 and the MNIC determination began at the concentration of 100%.

- MNIC determination:

24 hours after removal of the occlusive dressings, a slight erythema was recorded in three animals on the treated area at 100% and 50% and a slight erythema was recorded in two animals on the treated area at 25%.

In view of this result, the concentrations selected were 12.5% (MNIC) and 6.25% (1/2 MNIC) for the challenge phase.

Main study:

- Induction phase Group 2:

The induction phase was performed by intradermal injection at D0 with the test item at 6.25% in olive oil and by topical application at D7 with the test item at 100%.

No cutaneous reaction was recorded after the 1st induction phase. It was recorded a dryness in all animals (10/10), 24 hours after the removal of the occlusive patch during the 2nd induction.

- Induction phase Group 1:

The induction phase was performed by intradermal injection at D0 with olive oil and by topical application at D7 with liquid paraffin.

No cutaneous reaction was recorded during the induction phase.

- Challenge phase Groups 1 & 2:

The test item has been used diluted at 12.5% (MNIC) and 6.25% (1/2 MNIC) in liquid paraffin.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In view of the results, under these experimental conditions, the test item
NOREENAL must be classified R43 “May cause sensitisation by skin contact”, in
accordance with the criteria for classification, packaging and labelling of dangerous substances and
preparations of the E.E.C..
In accordance with the Globally Harmonized System (COM(2007)355 final), the test item must be
classified in category 1. The signal word “Warning” and hazard statement H317 “May cause an
allergic skin reaction” are required.
Executive summary:

The aim of the study was to evaluate the possible allergenic activity of the test item NOREENAL

after intradermic injection and topical administration in guinea pigs.

After induction (intradermic injection at 6.25% and topical application at 100%) of 10 Guinea Pigs of

treated group with the test item NOREENAL and a 10-day rest phase, the challenge phase,

under occlusive dressing for 24 hours, consisted to a single topical application of the test item diluted

at 12.5% and 6.25% in liquid paraffin. The experimental protocol was established according the

O.E.C.D. guideline n°406 dated July 17th, 1992 and the method B.6 of the E.E.C. n°96/54 dated July

30th, 1996.

It was recorded a slight to moderate erythema, in respectively 50% (5/10), 30% (3/10) and 10% (1/10)

of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the treated

area at 12.5%.

No cutaneous intolerance reaction was recorded in animals from the negative control group, on the

treated area at 12.5%.

It was noted a slight to moderate erythema in 10% (1/10) of the animals from the treated group, 24, 48

and 72 hours after the challenge phase, on the treated area at 6.25%.

No cutaneous intolerance reaction was recorded in animals from the negative control group, on the

treated area at 6.25%.

In conclusion, in view of these results, under these experimental conditions, the test item

NOREENAL must be classified R43 “May cause sensitisation by skin contact”, in

accordance with the criteria for classification, packaging and labelling of dangerous substances and

preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. This item must be characterised by

the symbol “Xi” and the warning label “Irritant”.

In accordance with the Globally Harmonized System (COM(2007)355 final), the test item must be

classified in category 1. The signal word “Warning” and hazard statement H317 “May cause an

allergic skin reaction” are required.