Registration Dossier

Administrative data

Description of key information

The study was carried out in accordance with the OECD Guideline No. 406, "Skin Sensitisation", The persistent responses in two experimental animals (total = 10) were considered indicative of sensitisation, since no such reactions were observed in the control animals. These results lead to the conclusion by the author that HEOX-CN has a mild sensitisation rate of 20 per cent according to the rating of allergenicity described by Kligman A. M. (1966). However, according to According to the Guidance to Regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, November 2012, the test substance is not classified. For a significant skin sensitizing effect, the EU CLP regulation (2012) requires redness in 30% or more of the guinea pigs tested at >1%  (W/V) intradermanl induction concentration in the Ginea Pig Maximization Test.  Therefore, HEOX-CN is considered to be a non-sensitizer to the skin.

Justification for selection of skin sensitisation endpoint:

Valid conclusion from a GLP study

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
before adoption of LLNA
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS

IN-LIFE DATES: From: 28 August 1995 To: 28 September, 1995

ANIMAL HUSBANDRY Conditions:
Dunkin Hartley Crl: (HA)BR albino gUinea pig (SPFquality), recognised by international guidelines as the recommended test system (e. g. OECD, EEC) from Charles River, Germany were used.
Age at start of study: Approx. 6 weeks
Body weight prior to start: Less than 500 grams
Idenification: Ear tag

A positive control experiment is performed once every six months as a sensitivity check of the test system. The most recent test is summarised in the Appendix of the study report.

Air-conditioned room with approximately 15 air changes per hour and the environment controlled with optimal conditions considered as being a temperature of 21°C and a relative humidity of 50%. Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity. Lighting was 1 2 hours artificial fluorescent light and 12 hours dark per day.

Accommodation:
Group housing of 2 animals, except for 1 control animal, per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands). The acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

Diet:
Free access to standard guinea pig diet, including ascorbic acid (16 00 mg/kg); L C 23-B, pellet diameter 4mm (Hope farms, Woerden, The Netherlands). Certificates of analysis were examined and retained in the NOTOX archives. In addition, hay (B.M.I., Helmond, The Netherlands) was provided once a week.
water:
Free access to tap water, diluted with decalcified water. Certificates of analysis were examined and retained in the NOTOX archives.

Dunkin Hartley Crl: (HA)BR albino gUinea pig (SPFquality), recognised by international guidelines as the recommended test system (e. g. OECD, EEC) .
Source : Charles River, Germany, were used.


Approx. 6 weeks
Less than 500 grams
Ear tag
A positive control experiment is performed once every
six months as a sensitivity check of the test system.
The most recent test is summarised in the Appendix.
Route:
other: intradermal and epidermal
Vehicle:
corn oil
Remarks:
Corn oil was selected based on a pretest performed at NOTOX. When required, the test substance formulations (w/w) were prepared prior to each treatment. No adjustment was made for specific gravity of vehicle. Homogeneity was obtained to visually acceptabl
Concentration / amount:
Induction: Intradermal injection: 2%; and epidermal: 50% concentration
Challenge: 5%, 10%, and 25% test substance concentration, by epidermal application
Second challenge: 25% test substance concentration
Route:
intradermal
Vehicle:
corn oil
Remarks:
Corn oil was selected based on a pretest performed at NOTOX. When required, the test substance formulations (w/w) were prepared prior to each treatment. No adjustment was made for specific gravity of vehicle. Homogeneity was obtained to visually acceptabl
Concentration / amount:
Induction: Intradermal injection: 2%; and epidermal: 50% concentration
Challenge: 5%, 10%, and 25% test substance concentration, by epidermal application
Second challenge: 25% test substance concentration
No. of animals per dose:
Experimental group : 10 females
Control group 5 females
Details on study design:
PRELIMINARY STUDY
Prior to the start of the Main study, the intradermal and epidermal irritancy of HEOX-CN was investigated to select test substance concentrations suitable for the induction and challenge phase of the Main Study. The procedures and techniques were identical to those used during the main study, unless otherwise mentioned. The animals were selected from stock and were between 5 and 9 weeks old.

Intradermal injections:
The undiluted test substance and a 50% concentration were injected in one animal into two injections sites (0.1 ml each) in the left and right clipped shoulder regions, respectively. A 25% and 5% concentration were injected in a similar manner in a second animal. The resulting dermal reactions were assessed 24 and 48 hours later for erythema (see scale below), necrosis and diameter of necrosis.

Epidermal application:
The undiluted test substance (0.5 ml) was applied epidermally on a clipped flank of one animal, using a Scotchpak-non-woven patch* (2.5 x 2.2 cm) on Micropore tape* (* Supplier, 3M, St. Paul, U.S.A) and held in place by Coban elastic bandage*. A 50% concentration was applied in a similar manner on a second animal.

Four concentrations of the test substance (undiluted, 50%, 25% and 10%, 0.05 ml each) were applied occlusively on a clipped flank of each of two other animals using Square chambers (v.d. Bend, Brielle, The Netherlands), mounted on Micropore tape and held in place by Coban elastic bandage.

After 24 hours, the dressings were removed and the skin cleaned of residual test substance. The treated skin-sites were assessed 24 and 48 hours after exposure according to the scale described below.

Additional testing:
Since no suitable test substance concentrations could be selected from the results obtained with the above treated animals, an additional animal was intradermally injected with a 1% and 0.5% concentration and another animal with a 0.2% and 0.1% concentration. All further procedures were similar to those described above .

Erythema and eschar formation:
No erythema . . . . . . . ............. . . . . . . . . . . . . . , ........................................................... 0
Slight erythema (barelyerceptible) . . . . . ........... . . . . . . . . . .. . .. . . . . . . . . . . . . ….. 1
Well-defined erythema . . . . . . . . . . ... . . . . . . . ........... . . . . . . . . .. . . . . . . . . . . . ….. 2
Moderate erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... . . . . . . . . . . . . . . . . . . 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) . 4

Oedema formation:
No oedema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................ . . . . . . .0
Slight oedema (barely perceptible) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ . .1
Well-defined oedema (edges of area well-defined by definite raising) . . . . . . . . . . . . . . . . . ..... …. 2
Moderate oedema (raised approximately 1 millimeter) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) . 4


MAIN STUDY
INDUCTION - EXPERIMENTAL ANIMALS
Day 1: Three pairs of intradermal injections (0.1 ml/site) were made in the clipped scapular region as follows:
A) Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.), 50:50 with water for injection (Ferensius AG, Bad Homburg, Germany).
B) The test substance at a 2% concentration.
C) The test substance, at twice the concentration used in (B), emulsified in a 50:50 mixture of Freunds' Complete Adjuvant.

Note: One of each pair was on each side of the midline and from cranial A) to caudal C).

Day 3: The dermal reactions caused by the intradermal injections were recorded.
Day 8: The clipped area between the injection sites was treated with 0.5 ml of a 50% test substance concentration using a Scotchpak-non-woven patch (2 x 4 cm) mounted on Micropore tape and secured with Coban elastic bandage.
Day 10: After 48 hours, the dressings were removed and the skin cleaned of residual test substance. Subsequently, all dermal reactions caused by theepidermal exposure were recorded, according the scale presented before.

INDUCTION - CONTROL ANIMALS
Day 1: Intradermal injections were made similar to the method described above with the omission of the test substance.
Day 3: The dermal reactions caused by the intradermal injections were recorded.
Day 8: Epidermal treatment as described for the experimental animals, but with the vehicle only.
Day 10: After 48 hours, the dressings were removed and the skin cleaned. Subsequently, all dermal reactions caused by the epidermal exposure were recorded, according the scale presented before.

CHALLENGE
Day 22: All animals were treated epidermally with 0.05 ml of each of a the following test substance concentrations, 25%, 10% and 5% and the vehicle on the clipped left flank, using Square chambers, attached to Micropore tape and secured with Coban elastic bandage.

Day 23: After 24 hours, the dressings were removed and the skin cleaned of residual test substance and vehicle. The treated sites were assessed for erythema and swelling 24 and 48 hours after removal of the dressings, using the numerical grading system described below. After the first reading, the test sites were clipped with an electric clipper.

No visible change . . . . . . . . . . . . . . . . . . .. . . . ... . ... . . . 0
Discrete or patchy erythema ..... ....... ................. 1
Moderate and confluent erythema ....... ..... .. ..... ...... 2
Moderate erythema and swelling . . . . ... . . . . . .. . . . . . . 3
Intense erythema and swelling . . . . . . . .. . . . . .. . . . . . . 4

RE-CHALLENGE
Day 29: To confirm the results of the first challenge, a second challenge was
conducted one week later.
All animals were treated epidermally with a 25% concentration and the vehicle (0.5 ml each) on the clipped right flank. The treatment was performed, using a Scotchpak-non-woven patches (2.5 x 2.2 cm) mounted on Plastic Medical tape (3M, St. Paul, USA) and secured with Micropore tape and subsequently Coban elastic bandage. After approximately 24 hours, the dressings were removed and the skin cleaned of residual test substance and vehicle.The treated sites were assessed in a similar manner as during the first challenge.

At the end of the study period all animals were killed by oxygen/carbon dioxide asphyxiation.
Challenge controls:
For the challenge and re-challenge conditions, please see the "details on study design" (above)
Positive control substance(s):
yes
Remarks:
alpha hexyl cinnaminicaldehyde
Positive control results:
A positive control experiment was carried out at regular intervals to check the reliability of the test system as used by NOTOX. The Dunkin Kart1ey guinea pig was checked for the sensitivity to ALPHA-HEXYLCINNAMICALDEHYDE, TECH. 85%. The study was
carried out in accordance with the DECO Guideline No. 406. The results lead to a sensitisation rate of 100 per cent.
Reading:
1st reading
Remarks on result:
other: see "Any other information"

RESULTS

PRELIMINARY STUDY

The results of the intradermal injections and epidermal exposures for the selection of suitable test substance concentrations for the main study are described in Table 1. Based on the results, the test substance concentrations for the Main Study were selected to be a 2% concentration for the intradermal induction and a 50% concentration for the epidermal induction exposure. A 25%, 10% and 5% concentration were selected for the challenge phase.

 

MAIN STUDY

Induction phase: The skin effects caused by the intradermal injections and epidermal exposure during the induction phase are given in Table 2.

 

Challenge phase:

First Challenge:

Skin reactions, consisting of grade 1, were observed in seven experimental animals in response to one or more of the three test substance concentrations, 24 hours after the challenge exposure. One response to a 10% concentration persisted at the following observation. Similar skin reactions were observed in one control animal in response to the 25% and 10% concentrations, 24 hours after the challenge exposure only.

 

Second challenge.

To confirm the results obtained in the first challenge, a second challenge was performed one week later with the 25% concentration and the vehicle.

 

Skin reactions, consisting of grade 1, were observed in nine experimental animals in response to the 25% test substance concentration, 24 hours after the challenge exposure. The responses in two animals persisted at the following observation. Scaliness was observed in one experimental animal. Skin reactions grade 1 were also observed in two control animals in response to the 25% concentration, 24 hours after the challenge exposure only.

 

Toxicity Symptoms I Mortality

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

 

Body Weights

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

CONCLUSION

The responses in four experimental animals were considered non-specific signs of irritation, since no reactions were observed to the highest concentration (i. e. 25%) in these animals. It could not be decided whether the responses in the three other animals were indicative of sensitisation, since similar responses were observed in a control animal. In the second challenge, similar responses were noted in both the experimental and control animals 24 hours after the challenge exposure. The persistent responses in two experimental animals were considered indicative of sensitisation, since no such reactions were observed in the control animals.

 

These results lead to a sensitisation rate of 20 per cent. Applying the rating of allergenicity described by Kligman A. M. (1966) on the results obtained in this test, HEOX-CN is considered to have mild sensitizing properties.

 

Based on these results and according to the EEC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC, 27th April 1993), HEDX-CN cannot be classified and has no obligatory labelling requirement. 

Interpretation of results:
not sensitising
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
These results lead to a low sensitisation rate of 20 per cent. Applying the rating of allergenicity described by Kligman A. M. (1966) on the results obtained in this test, HEOX-CN was interpretted to have had mild sensitizing properties. However, according to According to the Guidance to Regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, November 2012, the test substance is not classified. For significant skin sensitizing effect, the EU CLP regulation requires redness in 30% or more of the guinea pigs tested at >1% (W/V) intradermanl induction concentration in the Guinea Pig Maximization Test.
 
Executive summary:

Assessment for Contact Hypersensitivity to HEDX-CN in the Albino Guinea Pig (Maximization Test).

 

This GLP study was carried out in accordance with the DECD Guideline No. 406, "Skin Sensitisation", the EEC Directive 9 2/69/EEC, Part B. 6, " Skin Sensitisation' and based on the method described by Magnusson and Kligman, "Allergic contact Dermatitis in the Guinea Pig - Identification of contact Allergens".

 

The purpose of this study was to evaluate whether HEOX-CN induces contact hypersensitivity in guinea pigs after intradermal and epidermal exposure of the animals under the conditions described in this report. This study should provide a rational basis for potential sensitising risk assessment in man. The intradermal route of administration (first induction) was selected in order to obtain optimal contact between the test substance and elements of the immune system. The epidermal route of administration (second induction and challenge) was selected as it is a possible route of human exposure during manufacture, handling or use of the test substance. A positive control experimanet data were also provided in the report.

 

Test substance concentrations (in corn oil) selected for the Main study were based on the results of a preliminary study. In the Main study, ten experimental animals were intradermally injected with a 2% concentration and epidermally exposed to a 50% concentration. Five control animals were similarly treated, but with omission of the test substance. Two weeks after the epidermal application all animals were challenged with a 25%, 10% and 5% test substance concentration and the vehicle. To confirm the results obtained in the first challenge, a second challenge was performed one week later with the 25% concentration only and the vehicle. In both challenge phases, comparable reactions (grade 1 only) were observed in both the experimental and control animals. These reactions were observed mainly within 24 hours after the challenge exposure. In the second challenge, responses persisted for 48 hours in two experimental animals, whereas no such reactions were observed in the control animals. These responses were considered indicative of sensitization

 

These results lead to a sensitisation rate of 20 per cent. Applying the rating of allergenicity described by Kligman A. M. (1966) on the results obtained in this test, HEDX-CN is considered to have mild sensitizing properties.

 

Based on these results and according to the EEC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC, 27th April 1993), HEDX-CN cannot be classified and has no obligatory labelling requirement.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on test results, the substance is considered to be a non-sensitizer to the skin