Registration Dossier

Administrative data

Description of key information

Skin sensitisation: Non sensitiser in LLNA (Mouse).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November to 24 November 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
not specified
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species: Mouse, CBA strain, inbred, SPF-Quality. Recognised by the international guidelines as the recommended test system (e.g. OECD, EEC, EPA) Source: Charles River France, L'Arbresle Cedex, France
Number of animals: 20 females (four groups of five females each group) (nulliparous and non-pregnant).
Age and bodyweight: Young adult animals (approx. 9 weeks old) were selected. Bodyweight variation was +/- 20% of the sex mean.
Identification: Tail mark.
Control animals: The vehicle control animals were treated using the same vehicle and using the same procedures.
Reliability check: Similar procedures were used in the reliability test and in this study

Animal husbandry
Conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 18.6 - 21.0°C), a relative humidity of 30-70% (actual range: 40 - 63%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Accommodation: Individual housing in labelled Macrolon cages (type I; height 12.5 cm) containing purified sawdust as bedding material (SAWI, Jelu Werk, Rosenberg, Germany). Certificates of analysis were examined and then retained in the NOTOX archives. The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. Animals were group housed in polycarbonate cages (Macrolon II type; height 15 cm) during the acclimatisation period.
Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany).
Water: Free access to tap-water.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1% and if needed further lower concentrations using the same steps.
No. of animals per dose:
Five animals per dose concentration
Details on study design:
Preliminary irritation study
A preliminary irritation study was conducted as part of NOTOX project 385368 in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 3) at the highest.
A series of four test substance concentrations were tested, the highest concentration being the maximum concentration that could technically be applied. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1% and if needed further lower concentrations using the same steps. The test system, procedures and techniques were identical to those used during days 1 to 3 of the main study unless otherwise specified.
Four young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on two consecutive days. Approximately 4 hours after the last exposure, the skin was cleaned of residual test substance with water and the irritation was assessed. No necropsy was performed and no bodyweights were determined after termination.

Main study
Three groups of five animals were treated with three test substance concentrations respectively.
One group of five animals was treated with vehicle.
ALLOCATION
GROUP* INDUCTION
1 (1-5) Vehicle control Vehicle
2 (6-10) Experimental Lowest test substance concentration: 5%
3 (11-15) Experimental Intermediate test substance concentration: 50%
4 (16-20) Experimental Highest test substance concentration: 100%
* five females each group, animal numbers between brackets

INDUCTION - Days 1, 2 and 3
Experimental animals: The dorsum surface of both ears was epidermally treated (25 µl/ear) with the test substance concentration, approximately the same time each day.
Vehicle control animals: The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle was administered.
TREATMENT - Day 6:
All animals: Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (Amersham Pharmacia Biotech, NOTOX Substance 105624).
After approximately five hours, all animals were killed by intra peritoneal injection with an overdose of pentobarbital. 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 were recorded. The nodes were pooled for each animal in 3 ml PBS.

Tissue processing for radioactivity: A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 I'm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4° C. The DNA was precipitated with 3 ml 5% trichloroacetic acid (TCA) at 4° C for approximately 18 hours.
Precipitates were recovered by centrifugation at 200g for 10 minutes, resuspended in 1 ml TCA and transferred to 10 ml of Ultima Gold (Packard) as the scintillation fluid.

Radioactivity measurements: All radioactive measurements were performed using a Packard scintillation counter (1900TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The Packard 1900TR was programmed to automatically subtract background and convert CPM to DPM.

Observation
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Bodyweights: On days 1 (pre-treatment) and 6.
Irritation: On day 3 (3-4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

GRADING IRRITATION REACTIONS
Erythema and eschar formation:
No erythema: 0
Slight erythema (barely perceptible): 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 millimetre): 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure): 4


Interpretation: 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. For undiluted test substance, untreated animals are used to calculate the SI.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM (NIH publication; No 99-4494, February 1999).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A Stimulation Index (Si) is calculated for each group.
Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX. In this study, performed in August 2003, females of the CBA mouse strain (from Charles River France, L'Arbresle Cedex, France) were checked for the sensitivity to ALPHA-HEXYLCINNAMICALDEHYDE, TECH. 85%.
The females were approx. 7-8 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, the EC Directive 67/548/EC, Part B.42 and on the method described by ICCVAM (NIH publication: No 99-4494, February 1999). ALPHAHEXYLCINNAMICALDEHYDE, TECH. 85% (CAS no. 101-86-0) was fabricated under lot no. 10021HF (Aldrich Chemicals Co., Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1).

CONCLUSION
The SI values calculated for the substance concentrations 5, 10 and 25% were 2.5, 7.5 and 25.4 respectively.
An EC3 value of 5.5% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%.
Based on these results it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
The raw data, protocol and report from this study are kept in the NOTOX archives. The test described above was performed in accordance with NOTOX Standard Operating Procedures and the report was audited by the QA-unit.
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
100%

PRELIMINARY IRRITATION STUDY: Based on the results, the test highest test substance concentration selected for the main study was a 100% concentration.

 

MAIN STUDY

Induction phase: The skin effects seen after the third epidermal exposure are presented in the table.

Macroscopy: The majority of nodes were equal in size, except for the left node of animal no. 16 which was slightly enlarged when compared to the control nodes. No other macroscopic abnormalities of the nodes were noted.

Body Weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, wasconsidered not toxicologically significant.

Radioactivity Measurements:Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 50 and 100% were 192, 326 and 471 respectively.The mean DPM/animal value for the vehicle control group was 195.

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

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
HATCOL 3331 should not be regarded as a skin sensitiser.
Executive summary:

Assessment for Contact Hypersensitivity to HATCOL 3331 in the Mouse (Local Lymph Node Assay).

The study was carried out based on the guidelines described in: OECD, Section 4, Health Effects, No.429 (2002), Paris Cedex; EC, Council Directive 67/548/EEC, Annex IV C, B.42 (Draft) (2001); Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.2600. "Skin Sensitisation" 2003.

Test substance concentrations selected for the main study were based on the results of a preliminary study (NOTOX Project 385368).

In the main study, three groups of five experimental animals were epidermally exposed to a 5%, 50% and 100% concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.

After precipitating the DNA of the lymph node cells, radioactivity measurements were done.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 50 and 100% were 192, 326 and 471 respectively. The mean DPM/animal value for the vehicle control group was 195.

The SI values calculated for the substance concentrations 5, 50 and 100% were 1.0, 1.7 and 2.4 respectively.

These data showed a dose-response but there was no indication that the test substance could elicit a SI ≥ 3.

Based on these results and according to the recommendations made in the test guidelines (OECD NO.429, EC B.42 and EPA OPPTS 870.2600), HATCOL 3331 should not be regarded as a skin sensitiser.

Based on these results and according to the:

OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998), HATCOL 3331 does not have to be classified for sensitisation by skin contact.

EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), HATCOL 3331 does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 to 24 November 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
not specified
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species: Mouse CBA strain, inbred, SPF-Quality. Recognised by the international guidelines as the recommended test system (e.g. OECD, EEC, EPA). Source: Charles River France, L'Arbresle Cedex, France.
Number of animals: 15 females (three groups of five females each group) (nullparous and non-pregnant).
Age and bodyweight: Young adult animals (approx. 9 weeks old) were selected. Body weight variation was +/- 20% of the sex mean.
Identification: Tail mark.
Control animals: The results of the vehicle control animals in NOTOX Project 396877 were used as reference for the treated animals of this study. The vehicle control animals were treated using the same vehicle and procedures, and within the same time frame as this study.
Reliability check: The results of a reliability test performed not more than 6 months previously or 2 months afterwards. Similar procedures were used in the reliability test and in this study.

Conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 18.6- 21.0°C), a relative humidity of 30-70% (actual range: 40 - 63%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Accommodation: Individual housing in labelled Macrolon cages (type I; height 12.5 cm) containing purified sawdust as bedding material. The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. Animals were group housed in polycarbonate cages (Macrolon II type; height 15 cm) during the acclimatisation period.
Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany).
Water: Free access to tap-water.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 50% and 100%
No. of animals per dose:
Five females per each dose group.
Details on study design:
Preliminary irritation study: A preliminary irritation study (NOTOX project 385302) was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 3) at the highest.
A series of four test substance concentrations were tested, the highest concentration being the maximum concentration that could technically be applied. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1 % and if needed further lower concentrations using the same steps. The test system, procedures and techniques were identical to those used during days 1 to 3 of the main study unless otherwise specified.
Four young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on two consecutive days. Approximately 4 hours after the last exposure, the skin was cleaned of residual test substance with water and the irritation was assessed. No necropsy was performed and no bodyweights were determined after termination.

Main study: Three groups of five animals were treated with three test substance concentrations respectively.
ALLOCATION
GROUP* INDUCTION
1 (1-5) Experimental Lowest test substance concentration: 5%
2 (6-10) Experimental Intermediate test substance concentration: 50%
3 (11-15) Experimental Highest test substance concentration: 100%
*five females each group, animal number between brackets

INDUCTION - Days 1, 2 and 3
Experimental animals: The dorsum surface of both ears was epidermally treated (25 µl/ear) with the test substance concentration, approximately the same time each day.
TREATMENT - Day 6:
All animals: Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PSS) containing 20 µCi of 3H-methyl thymidine (Amersham Pharmacia Biotech, NOTOX Substance 105624).
After approximately five hours, all animals were killed by intra peritoneal Injection with an overdose of pentobarbital. 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 were recorded. The nodes were pooled for each animal in 3 ml PBS.

Tissue processing for radioactivity: A single cell suspension of lymph node cells (LNC) was prepared in PSS by gentle separation through stainless steel gauze (diameter 125 µm). LNC were washed twice with an excess of PSS by centrifugation at 200g for 10 minutes at 4° C. The DNA was precipitated with 3 ml 5% trichloroacetic acid (TCA) at 4° C for approximately 18 hours.
Precipitates were recovered by centrifugation at 200g for 10 minutes, resuspended in 1 ml TCA and transferred to 10 ml of Ultma Gold (Packard) as the scintillation fluid.

Radioactivity measurements: All radioactive measurements were performed using a Packard scintillation counter (1900TR). Counting time was to a statistical precision of:l 0.2% or a maximum of 5 minutes whichever comes first. The Packard 1900TR was programmed to automatically subtract background and convert CPM to DPM.

Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On days 1 (pre-treatment) and 6.
Irritation: On day 3 (3-4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded according to a numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Interpretation: 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. For undiluted test substance, untreated animals are used to calculate the SI.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM (NIH publication; No 99-494, February 1999).
The results were evaluated according to the OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998) and the EC criteria for classification and labelling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptations to technical progress and amendments of this Directive published In the Official Journal of the European Communities).
If possible, an EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not specified in the study report.
Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX. In this study, performed in August 2003, females of the CBA mouse strain (from Charles River France, L'Arbresle Cedex, France) were checked for the sensitivity to ALPHA-HEXLCINNAMICALDEHYDE, TECH. 85%.
The females were approx. 7-8 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, the EC Directive 67/54B/EC, Part 6.42 and on the method described by ICCVAM (NIH publication; No 99-4494, February 1999). ALPHAHEXLCINNAMICALDEHYDE, TECH. 85% (CAS no. 101-86-0) was fabricated under rot no. 10021 HF (Aldrich Chemicals Co., Germany). Concentrations used for this study were 5. 10 and 25% in Acetone:Olive oil (4:1).
Group* Treatment Induction Mean SI ± SD
DPM ± SD
1 Vehicle control Vehicle 245 ± 134 1.0
2 Experimental 5% test substance 623 ± 509 2.5 ± 1.0
3 Experimental 10% test substance 1840 ± 2212 7.5 ± 1.3
4 Experimental 25% test substance 6205 ± 2476 25.4 ± 0.7
*Groups 2, 3 and 4, four females each group. Group 1, three females.

CONCLUSION
The SI values calculated for the substance concentrations 5. 10 and 25% were 2.5. 7.5 and 25.4 respectively.
An EC3 value of 5.5% was calculated using linear Interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%.
Based on these results it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
The raw data, protocol and report from this study are kept in the NOTOX archives. The test described above was performed in accordance with NOTOX Standard Operating Procedures and the report was audited by the QA-unit.
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
2.6
Test group / Remarks:
100%

PRELIMINARY IRRITATION STUDY: Based on the results, the test highest test substance concentration selected for the main study was a 100% concentration.

 

MAIN STUDY

Macroscopy: The majority of nodes were equal in size, except for the nodes of animal nos. 6 (right node), 10 (left node), 14 (right node) and 15 (left node). No other macroscopic abnormalities of the nodes were noted.

Body Weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss noted for animal no. 12 was considered not toxicologically significant.

Radioactivity Measurements: Mean DPM/animal values for the experimental groups treated with test substanceconcentrations 5, 50 and 100% were 229, 184 and 513 respectively.

The mean DPM/animal value for the vehicle control group was 195 (NOTOX Project 396877).

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

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
HATCOL 3344 should nol be regarded as a skin sensitiser.
Executive summary:

Assessment for Contact Hypersensitivity to HATCOL 3344 in the Mouse (Local Lymph Node Assay).

The study was carried out based on the guidelines described in: OECD, Section 4, Health Effects, No.429 (2002), Paris Cedex; EC, Council Directive 67/548/EEC, Annex IV C, B.42 (Draft) (2001); Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.2600. “Skin Sensitisation” 2003.

Test substance concentrations selected for the main study were based on the results of a preliminary study (NOTOX project 385302).

In the main study, three groups of five experimental animals were epidermally exposed to a 5%, 50% and 100% concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive 011 (4:1 v/v)) (NOTOX Project 396877).

Three days after the last exposure, all animals were injected with 3H~methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.

After precipitating the DNA of the lymph node cells, radioactivity measurements were done.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 50 and 100% were 229, 184 and 513 respectively. The mean DPM/animal value for the vehicle control group was 195 (NOTOX Project 396877).

The SI values calculated for the substance concentrations 5, 50 and 100% were 1.2, 0.9 and 2.6 respectively.

Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), HATCOL 3344 should not be regarded as a skin sensitiser.

Based on these results and according to the:

- OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998), HATCOL 3344 does not have to be classified for sensitisation by skin contact.

- EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), HATCOL 3344 does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November 2003 to 08 December 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
not specified
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species: Mouse, CBA strain. inbred, SPF-Quality. Recognised by the international guidelines as the recommended test system (e.g. OECD, EEC, EPA) Source: Charles River France, L'Arbresle Cedex, France
Number of animals: 25 females (five groups of five females each group). (Nullparous and non-pregnant).
Age and bodyweight: Young adult animals were selected (approx. 9 weeks old (animal nos. 1-15) or 12 weeks old (animal nos. 16-25)). Body weight variation was +/- 20% of the sex mean.
Identification: Tail mark.
Control animals: The results of the vehicle control animals in NOTOX Project 396877 were used as reference for animal nos. 1-15. The vehicle control animals were treated using the same vehicle and procedures, and within the same time frame as this study.
Reliability check: The results of a reliability test performed not more than 6 months previously or 2 months afterwards are summarised in the Appendix. Similar procedures were used in the reliability test and in this study.

Conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 18.1 - 22.3°C), a relative humidity of 30-70% (actual range: 16 - 69%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Accommodation: Individual housing in labelled Macrolon cages (type I; height 12.5 cm) containing purified sawdust as bedding material. The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. Animals were group housed in polycarbonate cages (Macrolon II type; height 15 cm) during the acclimatisation period.
Diet: Free access to standard pelleted laboratory animal diet.
Water: Free access to tap-water.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Lowest test substance concentration: 5%
Intermediate test substance concentration: 50%
Highest test substance concentration: 100%
No. of animals per dose:
Five females each dose group
Details on study design:
Preliminary irritation study
A preliminary irritation study (NOTOX project 366738) was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 3) at the highest.
A series of four test substance concentrations were tested, the highest concentration being the maximum concentration that could technically be applied. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1% and if needed further lower concentrations using the same steps. The test system, procedures and techniques were identical to those used during days 1 to 3 of the main study unless otherwise specified.
Four young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on two consecutive days. Approximately 4 hours after the last exposure, the skin was cleaned of residual test substance with water and the irritation was assessed. No necropsy was performed and no bodyweights were determined after termination.

INDUCTION - Days 1, 2 and 3
Experimental animals: The dorsum surface of both ears was epidermally treated (25 µl/ear) with the test substance concentration, approximately the same time each day.
Vehicle control animals: The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle was administered.
TREATMENT - Day 6:
All animals: Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) containing 20 µCI of 3H-methyl thymidine.
After approximately five hours, all animals were killed by intra peritoneal injection with an overdose of pentobarbital. 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 were recorded. The nodes were pooled for each animal in 3 ml PBS (nodes were processed separately for animal no. 6 since the right node was extremely enlarged).

Tissue processing for radioactivity
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 200gfor 10 minutes at 4°C. The DNA was precipitated with 3 ml 5% trichloroacetic acid (TCA) at 4°C for approximately 18 hours.
Precipitates were recovered by centrifugation at 200g for 10 minutes, resuspended in 1 ml TCA and transferred to 10 ml of Ultima Gold (Packard) as the scintillation fluid.

Radioactivity measurements
All radioactive measurements were performed using a Packard scintillation counter (1900TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The Packard 1900TR was programmed to automatically subtract background and convert CPM to DPM.

Observation
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On days 1 (pre-treatment) and 6.
Irritation: On day 3 (3-4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of another (local) effects were recorded.

Interpretation
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. For undiluted test substance, untreated animals are used to calculate the SI.
If the results Indicate a SI ≥ 3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM (NIH publication; No 99-494, February 1999).
The results were evaluated according to the DECO Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998) and the EC criteria for classification and labelling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptations to technical progress and amendments of this Directive published in the Official Journal of the European Communities).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
An EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation.
Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX. In this study, performed in August 2003, females of the CBA mouse strain (from Charles River France, L'Arbresle Cedex,
France) were checked for the sensitivity to ALPHA-HEXLCINNAMICALDEHYDE, TECH. 85%.
The females were approx. 7-8 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, the EC Directive 67/548/EC, Part B.42 and on the method described by ICCVAM (NIH publication; No 99-4494, February 1999). ALPHA-HEXLCINNAMICALDEHYDE, TECH. 85% (CAS no. 101-86-0) was fabricated under lot no. 10021 HF (Aldrich Chemicals Co., Germany). Concentrations used for this study were 5, 10 and 25% in Acetone:Olive oil (4:1).

CONCLUSION
The S1 values calculated for the substance concentrations 5, 10 and 25% were 2.5, 7.5 and 25.4 respectively.
An EC3 value of 5.5% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%.
Based on these results it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
100%

Grading of Skin Reactions after Epidermal Exposure

Animal number

Body weights (g)

Conc.%

Dorsal surface ear (Day 2)

Left

Right

Erythema

Oedema

Erythema

Oedema

1

21

100

2

0

2

0

2

24

50

2

0

1

0

3

25

25

2

0

1

0

4

22

10

1

0

2

0

Vehicle: Acetone/Corn oil (4:1 v/v)

 

Skin reactions after 3rdinduction, Body weights and Relative Size Nodes

Group treatment

An no

Day 1

Day 3

Day 6

BW (g)

Skin grading dorsal surface ear

BW (g)

Size nodes

Left

Right

Left

Right

Erythema

Oedema

Erythema

Oedema

Vehicle* Control**

 

23

0

0

0

0

22

+

+

 

19

0

0

0

0

19

+

+

 

21

0

0

0

0

21

+

+

 

22

0

0

0

0

23

+

+

 

22

0

0

0

0

21

+

+

1

5%

 test substance

1

22

0

0

0

0

22

+

+

2

22

0

0

0

0

23

+

+

3

20

0

0

0

0

20

+

+

4

20

0

0

0

0

20

+

+

5

20

0

0

0

0

21

+

+

2

50%

test substance

6

20

0

0

0

0

21

+

+++++

7

20

0

0

0

0

22

+

+

8

19

0

0

0

0

19

+

+

# 9

20

0

0

0

0

20

+

+

10

22

0

0

0

0

22

+

+

3

100%

test substance

11

22

0

0

0

0

24

+

+

12

20

0

0

0

0

21

+

+++

13

20

0

0

0

0

20

+

+

14

21

0

0

0

0

21

+

+

15

21

0

0

0

0

22

+

+

4 Vehicle* Control

16

20

0

0

0

0

19

+

+

17

24

0

0

0

0

23

+

+

18

26

0

0

0

0

25

+

+

19

23

0

0

0

0

22

+

+

$20

23

0

0

0

0

22

+

+

5

100%

test substance

21

21

0

0

0

0

21

+

+

22

25

0

0

0

0

24

+

+

23

23

0

0

0

0

22

+

+

24

25

0

0

0

0

24

+

+

25

23

0

0

0

0

23

+

+

Legends: BW = Body weight; Relative size nodes (-: reduced, +: enlarged)

# Right Mandibular lymph node enlarged

$ Red discolouration of right auricular lymph node

* Vehicle: Acetone/Olive oil (4:1 v/v)

** Determined under NOTOX project 396877

Radioactivity measurements (individual animals)

Group

Animal

Treatment

Induction

DPM/animal

SI/animal

 

 

Vehicle control (NOTOX project 396877)

Acetone/Olive oil (4:1 v/v)

101

-

104

-

131

-

447

-

194

-

1

1

Experimental

5% test substance

187

1.0

2

73

0.4

3

472

2.4

4

120

0.6

5

157

0.8

2

6

Experimental

50% test substance

#

-

7

365

1.9

8

353

1.8

9

39

0.2

10

164

0.8

3

11

Experimental

100% test substance

353

1.8

12

##

-

13

780

4.0

14

637

3.3

15

609

3.1

4

16

Vehicle control

Acetone/Olive oil (4:1 v/v)

270

-

17

307

-

18

400

-

19

722

-

20

479

-

5

21

Experimental

100% test substance

951

2.2

22

809

1.9

23

607

1.4

24

983

2.3

25

746

1.7

#71 (left node), 6068 (right node): value rejected; one node found to be extremely enlarged

## 2874: value rejected; one node found to be enlarged

 

Calculation of Stimulation Index (SI)

Group

Treatment

Induction

Mean

Mean

1

Experimental

5% test substance

202±157

1.0± 1.1

2

Experimental

50% test substance

230± 157

1.2± 1.0

3

Experimental

100% test substance

595± 178

3.0± 0.8

*

Vehicle control

Acetone/Olive oil (4:1 v/v)

195± 146

1.0

4

Vehicle control

Acetone/Olive oil (4:1 v/v)

436± 180

1.0

5

Experimental

100% test substance

819± 154

1.9± 0.5

3 + 5

Experimental

100% test substance

-

2.4± 0.9#

*Determined under NOTOX project 396877

#Calculated based on individual SI values. Individual SI values calculated by dividing the total count (DPM) by its concurrent mean control DPM value.

 

ASSESSMENT OF CONTACT HYPERSENSITIVITYTO ALPHA-HEXYLCINNAMIC ALDEHYDE, TECH., 85% IN THE MOUSE (LOCAL LYMPH NODE ASSAY)A RELIABILITY CHECK

Group*

Treatment

Induction

Mean

SI ± SD

DPM ± SD

1

Vehicle control

Vehicle

245 ± 134

1.0

2

Experimental

5% test substance

623 ± 509

2.5 ± 1.0

3

Experimental

10% test substance

1840 ± 2212

7.5 ± 1.3

4

Experimental

25% test substance

6205 ± 2476

25.4 ± 0.7

Groups 2, 3 and 4, four females each group, group 1, three females

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Hatcol 5236 should not be regarded as a skin sensitser.
Executive summary:

Assessment for Contact Hypersensitivity to HATCOL 5236 in the Mouse (Local Lymph Node Assay).

The study was carried out based on the guidelines described in: OECD, Section 4. Health Effects, No.429 (2002), Paris Cedex; EC, Council Directive 67/548/EEC, Annex IV C, B.42 (Draft) (2001); Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS870.2600. “Skin Sensitisation" 2003.

Test substance concentrations selected for the main study were based on the results of a preliminary study (Notox project 366738).

In the main study, initially three groups of five experimental animals were epidermally exposed to a 5%, 50% and 100% concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone:Olive oil (4:1 v/v)) (NOTOX Project 396877). An additional group of five animals was exposed to a 100% concentration (with a concurrent vehicle control group) to confirm the results in the first set of animals at this concentration.

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.

After precipitating the DNA of the lymph node cells, radioactivity measurements were done.

Mean DPM/animal values for the first set of experimental groups treated with test substance concentrations 5, 50 and 100% were 202, 230 and 595 respectively. The mean DPM/animal value for the vehicle control group was 195 (NOTOX Project 396877).

The mean DPM/animal value for additional group treated with the 100% test substance concentration was 819, with a mean DPM/animal value of 436 for the concurrent control group.

The SI values calculated for the substance concentrations 5, 50 and 100% were 1.0, 1.2 and 2.4 (overall mean SI).

Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), Hatcol 5236 should not be regarded as a skin sensitiser.

Based on these results and according to the:

OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998), Hatcol 5236 does not have to be classified for sensitisation by skin contact.

EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), Hatcol 5236 does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
08 Oct 1996 - 21 Jan 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Magnussen and Kligmann Method
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Available study data +12 years old. Used for supporting ionfirmation only.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: HRP inc. and Charles River Laboratories, USA- Age at study initiation: 5-7 weeks- Weight at study initiation: 391- 489 g (males); 366 - 480 g (females)- Housing: Individually housed in suspended, stainless steel cages with wire mesh bottoms- Diet: Agway Prolab Purina Guinea Pig Diet; ad libitum- Water: ad libitum- Acclimation period: 15 daysENVIRONMENTAL CONDITIONS- Temperature and humidity was controlled daily- Photoperiod (hrs dark / hrs light): 12 / 12 h
Route:
intradermal and epicutaneous
Vehicle:
propylene glycol
Concentration / amount:
Induction: Intradermal injection 5%, epicutaneous application 100%Challenge: 100%
Route:
epicutaneous, open
Vehicle:
propylene glycol
Concentration / amount:
Induction: Intradermal injection 5%, epicutaneous application 100%Challenge: 100%
No. of animals per dose:
20
Details on study design:
RANGE FINDING TESTS: Yes (Intradermal: October 1st 1996 - October 3rd 1996; Topical: October 1st 1996 - October 4th 1996)Two animals were pre-treated with two intradermal injections of FCA/water emulsion (1:1) approx. one week prior to test material administration. Animals were administered 0.1 mL of the test substance (5% w/v) in propylene glycol via injection on either side of the spinal column and observed for dermal irritation 24 h and 48 h after injection.For the topical range-finding study animals (3 per sex) were dosed with 0.1 mL of four different concentrations (25%, 50%, 75% and 100% in ethyl alcohol) at different sites, two on either side of the column. Treatment was for 24 h under occlusive conditions and observed for dermal irritation 24 h and 48 h afterwards.MAIN STUDYA. INDUCTION EXPOSURE- No. of exposures: 2- Test groups: 1- Control group: 2 (positive and irritation control)- Site: A row of three injections was made on each side in the shoulder region.- Frequency of applications: Days 1 and 8- Duration: 25 days- Concentrations: Intradermal induction: 5%, Topical induction: 100%B. CHALLENGE EXPOSURE- No. of exposures: 1- Day(s) of challenge: 14 d after last induction application- Exposure period: 24 h- Test groups: 1- Control group: 2 (positive and irritation control)- Site: Flank- Concentrations: 100%- Evaluation (hr after challenge): 24 and 48 h after removal of the patches
Positive control substance(s):
yes
Remarks:
Hexylcinnamic aldehyde (HCA)
Positive control results:
Treatment with 50% HCA resulted in 30% positive response.Treatment with 100% HCA resulted in 20% positive response (not sensitising, by definition). This lower effect might be due to decreased dermal absorption at high substance concentration.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
100%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 100%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 50%. No with. + reactions: 3.0. Total no. in groups: 10.0.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 1.0. Total no. in groups: 20.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
100%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 100%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 50%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
CLP: not classifiedDSD: not classified
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
31 March 2014 to 05 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species & Strain: Mouse; CBA/JcrJustification of Species: The mouse is species of choice for a local lymph node assay to provide information on which human hazard can be judged.Source: Harlan Sprague-Dawley; Indianapolis, INQuantity & Sex: 5 females per each Test group & 5 females in each Control group (all nulliparous & non-pregnant)Quarantine Period: 5 daysDate Born/Date Received: 21 Feb 14 / 17 Apr 14Anima/Group Identification: Tail marking / Cage cardWeights on Initial Dose Day: 20.4-26.3 gCage Type: Polycarbonate boxes with beddingHousing: 1 - 5 per cageEnvironmental Controls: Set to Maintain: Temperature: 22° ± 3°C; Relative humidity: 30 - 70%; 12-hour light/dark cycle; 10+ air changes per hourTemperature/Rel. Humidity: 19-23°C/52-84%Food: PMI Feeds Inc.™ Formulab #5008; available ad libitumWater: Municipal water supply analyzed by TCEQ Water Utilities Division; available ad libitum from automatic water systemAnimal husbandry and housing at STILLMEADOW, Inc. comply with standards outlined in "Guide for the Care and Use of Laboratory Animals" (NRC Publ.). No contaminants were expected to have been present in feed or water that would have interfered with or affected results of the study.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Each Test animal in its group received an open application of 25 µL of appropriate dilution (25 or 50%) of test substance, or 100% test substance undiluted.
No. of animals per dose:
5 mice per dose group
Details on study design:
Test Substance Preparation and Administration: Healthy mice were released from quarantine prior to testing. Five females were selected for each of three Test groups (Groups I- III). On Days 1, 2 and 3, each Test animal in its group received an open application of 25 µL of appropriate dilution (25 or 50%) of test substance, or 100% test substance undiluted, to the dorsum of both ears. The Vehicle Control group (5 females) was treated the same way as test animals, but with vehicle alone instead of test substance. The Positive Control group (5 females) was treated with alpha-hexylcinnamaldehyde as received. All Test and Control animals were given a two-day rest period on Days 4 and 5.Injection of Tritiated Methvl-Thymidine: On Day 6 of the study, all Test and Control animals were injected in the tail vein with 250 µL of 0.01 M phosphate-buffered saline (PBS; Sigma, Lot 081M8207, Exp Nov 2021), pH 7.4 at 25°C per manufacturer, containing 20 µCi of [methyl-3H) Thymidine (PerkinElmer, Lot 201404, Exp Apr 2015). Five hours after injection, animals were sacrificed with an overdose of CO2, the draining auricular lymph nodes excised and pairs from each individual animal processed.Suspension Preparation and DPM Determination: A single cell suspension was prepared by gentle mechanical disintegration through 200 mesh stainless steel gauze. Cells were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA; Ricca Chemical, Lot 2403 813, Exp Mar 20 15) at 4°C for 18 hours. The pellets were resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. Incorporation of tritiated thymidine was measured by liquid scintillation counting as disintegrations per minute (DPM) from paired lymph nodes of each animal, and mean DPM/animal was calculated for each group.Body Weights and Observations: Individual body weights were recorded on Day 1 prior to dosing, and Day 6, prior to injection. All Test and Control animals were observed daily for clinical signs of toxicity and any signs of excessive irritation at the test site.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not specified in the study report.
Positive control results:
Reported in table form - detailed under Any other information
Key result
Parameter:
SI
Remarks on result:
other: H2925 (CAS No. 68130-53-0) produced a stimulation index < 3 in all groups of Test animals.
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Reported in table form - detailed under Any other information

Stimulation Index or Test/Vehicle Control Ration derived for each Test group based on group mean DPM is as follows.

Animal Group

Test Substance Concentration

Average Count per Mouse

Number if Mice in Group

Test/Vehicle Control Ratio

Vehicle Control

NA

699

5

NA

Test Group I

25%

1173

5

1.7

Test Group II

50%

993

5

1.4

Test Group III

100%

917

5

1.3

Positive Control

NA

7696

5

11.0*

NA – Not applicable; *-Positive Control used to confirm animal sensitization potential and validate procedures.

 

TABLE 1 – Body Weights and DPM Counts

Test Substance: H2925 (CAS No. 68130-53-0)

Skin Sensitization: Local Lymph Node Assay in Mice

Animal No. (in Group)

Day of Study

DPM Count

Day 1 Wts.

Day 6 Wts.

Vehicle Control Group

1

23.5

24.3

967

2

24.2

25.0

1143

3

23.4

23.7

751

4

23.3

23.8

355

5

22.9

23.2

279

Test Group I – 25% concentration

1

26.3

27.3

811

2

24.8

24.9

664

3

21.5

22.6

1254

4

22.5

23.3

1461

5

25.4

25.9

1676

Test Group II – 50% concentration

1

25.2

25.6

1963

2

22.6

24.1

814

3

20.4

21.1

628

4

23.0

23.5

732

5

24.0

24.3

827

Test Group III – 100% concentration

1

24.1

24.4

836

2

23.2

24.0

615

3

24.9

25.1

525

4

22.1

23.4

673

5

24.5

25.2

1935

Positive Control Group

1

22.2

22.4

6743

2

22.7

23.4

1542

3

23.7

23.8

859

4

22.3

22.1

20841

5

23.8

23.9

8493

Note: Body weights are in grams.

 

TABLE 2 – Observations of Clinical Signs

Test Substance: H2925 (CAS No. 68130-53-0)

Skin Sensitization: Local Lymph Node Assay in Mice

Vehicle Control

 

Day

Reaction and Severity

1

2

3

4

5

6

Appeared normal at each observation

 

 

 

 

 

 

Test Group I – 25% concentration

 

Day

Reaction and Severity

1

2

3

4

5

6

Appeared normal at each observation

 

 

 

 

 

 

Test Group II – 50% concentration

 

Day

Reaction and Severity

1

2

3

4

5

6

Appeared normal at each observation

 

 

 

 

 

 

Test Group III – 100% concentration

 

Day

Reaction and Severity

1

2

3

4

5

6

Appeared normal at each observation

 

 

 

 

 

 

Positive Control

 

Day

Reaction and Severity

1

2

3

4

5

6

Appeared normal at each observation

 

 

 

 

 

 

 

Interpretation of results:
not sensitising
Remarks:
Migrated informationCriteria used for interpretation of results: EU
Conclusions:
H2925 (CAS No. 68130-53-0) produced a stimulation index < 3 in all groups of Test animals, and is not therefore considered a sensitizer (defined as producing a positive response).
Executive summary:

A skin sensitization study was conducted on 3 groups of 5 female mice to determine if test substance H2925 (CAS No. 68130-53-0) possesses a significant potential to cause skin sensitization. Five females were assigned to each of three groups, designated Groups I -III. Test groups were treated with an appropriate dilution (25 or 50%) in 4: 1 acetone: olive oil, or 100% test substance. Each animal received 25 µltothe dorsum of each ear. Animals were treated once daily for three days. After a two-day rest period, all animals were injected with tritiated methyl-thymidine in the tail vein. Five hours later, animals were sacrificed, and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A Vehicle Control group of five females was run concurrently, treated in the same manner with vehicle only instead of test substance or dilution. A Positive Control group of five females was also run concurrently, treated with alpha-hexylcinnamaldehyde as received.

The test substance produced a stimulation index < 3 in all groups of Test animals, and is not therefore considered a sensitizer (defined as producing a positive response).

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Composition of test substance unclear.
Principles of method if other than guideline:
Local Lymph Node Assay was performed in mice to investigate the skin sensitizing potential of the test substance.
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Vehicle:
other: Acetone
Concentration:
3%, 10% and 30%
No. of animals per dose:
4
Details on study design:
MAIN STUDY
- Name of test method: ß-scintillation counting
- Criteria used to consider a positive response:
1. The increase in isotope incorporation for at least one concentration tested must be three-fold or more compared to the control (vehicle treated) mice.
2. The data generated must be compatible with the biological dose response.

TREATMENT PREPARATION AND ADMINISTRATION:
Samples were administered to the dorsum of both ears using a micro-pipette.
Groups of 4 female mice were dosed with 25 µl of either vehicle (acetone) or a 30%, 10% or 3% preparation of the test item on three consecutive days on the dorsum of both ears. Five days after initial dosing, the animals received approx. 20 µCi of 3H-methyl thymidine, were sacrificed 5 h later and radioactive counts/lymph node were measured.
Key result
Parameter:
SI
Remarks on result:
other: The stimulation index was 0.45 for the 3% application, 2.05 for 10% and 1.21 for the 30% application.
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: No significant increase in isotope incorporation was detected after repeated application. The Cpm (Counts per minute) was 0.0022 Cpm and 0.0047 for the vehicle controls and 0.001, 0.0045 and 0.0057 for 3%, 10% and 30%, respectively.

Table 1: Results of the ß-scintillation counting

Test Concentration

Cpm/Lymph Node (x 10-2)

Test/Control Ratio

Vehicle

0.22

-

3% w/v

0.10

0.45

10% w/v

0.45

2.05

Vehicle

0.47

-

30% w/v

0.57

1.21

Interpretation of results:
other: inconclusive
Remarks:
Criteria used for interpretation of results: EU
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Significant methological deficiencies (only basic study data reported, no positive control, only 2 concentrations tested, test substance not defined).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
(only basic study data reported, no positive control, only 2 concentrations tested, test substance not defined)
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
No Data
Vehicle:
other: Acetone
Concentration:
3% or 10%
No. of animals per dose:
4
Details on study design:
MAIN STUDY
- Name of test method: ß-scintillation counting
- Criteria used to consider a positive response:
1. The increase in isotope incorporation for at least one concentration tested must be three-fold or more compared to the control (vehicle treated) mice.
2. The data generated must be compatible with the biological dose response.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of 4 female mice were dosed with 25 µl of either vehicle (acetone) or a 10% or 3% preparation of the test item on three consecutive days on the dorsum of both ears. Five days after initial dosing, the animals received approx. 20 µCi of 3H-methyl thymidine, were sacrificed 5 h later and radioactive counts/lymph node were measured.
Key result
Parameter:
SI
Remarks on result:
other: The stimulation index was 3.13 for the 3% application and 6.87 for the 10% application.
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: A significant increase in isotope incorporation was detected after repeated application. The Cpm (Counts per minute) increased dose-dependently from 0.0015 Cpm (Vehicle) to 0.0047 and 0.0103 for 3% and 10%, respectively.

Table 1: Results of the ß-scintillation counting

Test Concentration

Cpm/Lymph Node (x 10-2)

Test/Control Ratio

Vehicle

0.15

-

3% w/v

0.47

3.13

10% w/v

1.03

6.87

The increase in radioactive counts/lymph node seen between vehicle and 3% and 10% of the test compound implies a sensitizing potential. Only two concentrations were tested preventing a conclusive interpretation.

Interpretation of results:
other: inconclusive
Remarks:
Criteria used for interpretation of results: EU
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only short summary available (no data on test substance purity).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Only summary provided, no data on test substance purity
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
other: No Data
Strain:
not specified
Sex:
not specified
Vehicle:
not specified
Concentration:
1%, 3% and 10%
No. of animals per dose:
No Data
Key result
Parameter:
SI
Remarks on result:
other: The stimulation index was 1.3 for the 1% application, 1.19 for 3% and 1.54 for the 10% application.
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: No significant increase in isotope incorporation was detected after repeated application. The Cpm (Counts per minute)/Lymph node was 0.011 Cpm for the vehicle control and 0.0143, 0.0131 and 0.017 for 1%, 3% and 10%, respectively.

Table 1: Results of the ß-scintillation counting

Test Concentration

Cpm/Lymph Node (x 10-2)

Test/Control Ratio

Vehicle

1.1

-

1% w/v

1.43

1.3

30% w/v

1.31

1.19

10% w/v

1.7

1.54

The test substance is unlikely to be a sensitiser under the conditions of the test.

Interpretation of results:
other: inconclusive
Remarks:
Criteria used for interpretation of results: EU
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (no information on purity of the test material; both flanks were exposed during challenge, no reliability check done, no positive control used. Method given in very summarized form).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
no information on purity of the test material; both flanks were exposed during challenge, no reliability check done, no positive control used. Method given in very summarized form.
GLP compliance:
not specified
Type of study:
Buehler test
Species:
guinea pig
Strain:
other: Albino
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 344 - 441 g
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Induction: 100%
Challenge: 30% and 100%
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Induction: 100%
Challenge: 30% and 100%
No. of animals per dose:
20 (10 for the controls)
Details on study design:
RANGE FINDING TESTS: No Data

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: Undiluted test sample
- Control group: not stated
- Site: scapular region
- Frequency of applications: 7 d interval
- Duration: 14 d
- Concentrations: undiluted

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 28, 14 d after final induction
- Exposure period: 6 h
- Test groups: undiluted (100%) and 30% (w/v in corn oil)
- Control group: undiluted (100%) and 30% (w/v in corn oil)
- Site: undiluted (100%) on left flank and 30% on right flank
- Concentrations: undiluted (100%) and 30% (w/v in corn oil)
- Evaluation (hr after challenge): 24 h and 48 h
Challenge controls:
No
Positive control substance(s):
no
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
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: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
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: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0.
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified
Endpoint:
skin sensitisation
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
QSAR prediction: migrated from IUCLID 5.6
Guideline:
other: REACH guidance on QSARs R.6, May/July 2008
Principles of method if other than guideline:
(Q)SAR conducted with OECD Application Toolbox; Version 1.1.02
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

HATCOL 3331

Assessment for Contact Hypersensitivity to HATCOL 3331 in the Mouse (Local Lymph Node Assay).

Test substance concentrations selected for the main study were based on the results of a preliminary study (NOTOX Project 385368).

In the main study, three groups of five experimental animals were epidermally exposed to a 5%, 50% and 100% concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).

Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.

After precipitating the DNA of the lymph node cells, radioactivity measurements were done.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 50 and 100% were 192, 326 and 471 respectively. The mean DPM/animal value for the vehicle control group was 195.

The SI values calculated for the substance concentrations 5, 50 and 100% were 1.0, 1.7 and 2.4 respectively.

These data showed a dose-response but there was no indication that the test substance could elicit a SI ≥ 3.

Based on these results and according to the recommendations made in the test guidelines (OECD NO.429, EC B.42 and EPA OPPTS 870.2600), HATCOL 3331 should not be regarded as a skin sensitiser.

Based on these results HATCOL 3331 does not have to be classified for sensitisation by skin contact.

 

HATCOL 3344

Assessment for Contact Hypersensitivity to HATCOL 3344 in the Mouse (Local Lymph Node Assay).

Test substance concentrations selected for the main study were based on the results of a preliminary study (NOTOX project 385302).

In the main study, three groups of five experimental animals were epidermally exposed to a 5%, 50% and 100% concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive 011 (4:1 v/v)) (NOTOX Project 396877).

Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.

After precipitating the DNA of the lymph node cells, radioactivity measurements were done.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 50 and 100% were 229, 184 and 513 respectively. The mean DPM/animal value for the vehicle control group was 195 (NOTOX Project 396877).

The SI values calculated for the substance concentrations 5, 50 and 100% were 1.2, 0.9 and 2.6 respectively.

Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), HATCOL 3344 should not be regarded as a skin sensitiser.

Based on these results HATCOL 3344 does not have to be classified for sensitisation by skin contact.

 

HATCOL 5236

Assessment for Contact Hypersensitivity to HATCOL 5236 in the Mouse (Local Lymph Node Assay).

Test substance concentrations selected for the main study were based on the results of a preliminary study (Notox project 366738).

In the main study, initially three groups of five experimental animals were epidermally exposed to a 5%, 50% and 100% concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone:Olive oil (4:1 v/v)) (NOTOX Project 396877). An additional group of five animals was exposed to a 100% concentration (with a concurrent vehicle control group) to confirm the results in the first set of animals at this concentration.

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.

After precipitating the DNA of the lymph node cells, radioactivity measurements were done.

Mean DPM/animal values for the first set of experimental groups treated with test substance concentrations 5, 50 and 100% were 202, 230 and 595 respectively. The mean DPM/animal value for the vehicle control group was 195 (NOTOX Project 396877).

The mean DPM/animal value for additional group treated with the 100% test substance concentration was 819, with a mean DPM/animal value of 436 for the concurrent control group.

The SI values calculated for the substance concentrations 5, 50 and 100% were 1.0, 1.2 and 2.4 (overall mean SI).

Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), Hatcol 5236 should not be regarded as a skin sensitiser.

Based on these results Hatcol 5236 does not have to be classified for sensitisation by skin contact.

HATCOL 1510

In a LLNA conducted in accordance with OECD Guideline 429, the test substance produced a stimulation index < 3 in all groups of test animals, therefore the test substance is not considered a be a skin sensitiser.

 

CAS 11138-60-6

A guinea pig maximisation test was conducted with, decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS No. 11138-60-6) in accordance with OECD Guideline 406. A total of 20 male and female Dunkin-Harley guinea pigs were treated with the test substance and compared with 10 negative control animals. The sensitivity of the animal strain was tested using Hexylcinnamic aldehyde (HCA) as positive control substance. A 5% dilution of the test substance in propylene glycol was used for intradermal induction and 100% used for epidermal induction of the scapular region on days 1 and 8 .Fourteen days after the last induction treatment, all animals were challenged epicutaneously with the undiluted test substance. Twenty-four hours after challenge one out of twenty animals showed a skin reaction compared to two out of ten for the positive control. Forty-eight hours after challenge exposure all skin examination scores were zero in all test animals. Under the conditions of this test, the read-across substance was not classified as a sensitiser.

HATCOL 1765

Four studies were conducted with Fatty acids, C5-10, esters with pentraerythritol (CAS 68424-31-7) according to OECD Guideline 406 (Buehler Test) and OECD Guideline 429 (Local Lymph Node Assay). In the first test, the skin sensitisation potential of the test substance was evaluated in guinea pigs with a Buehler test for (Lees, 1991a). 20 male albino guinea pigs were treated with the test substance and compared with 10 control animals. Three epidermal inductions were performed with 100 % test substance in weekly intervals for 6 hours under occlusive conditions. 14 days after the last induction treatment, all animals were challenged for 6 hours epicutaneously with 100% (left shorn flank) and 30% (right shorn flank) test substance (diluted in corn oil) under occlusive conditions. Animals were evaluated for skin reactions 24 and 48 h after challenge. No signs for irritation or sensitisation were observed during induction and challenge of the animals.

This result is supported by the findings of a Local Lymph Node Assay (Bugg, 1992). Only a short summary with no details on the study protocol or data interpretation was given. Nevertheless, test substance concentrations of 1%, 3%, and 10% indicated that the test substance was not a sensitizer under the conditions chosen.

Furthermore, another Local Lymph Node Assay was conducted (Robinson, 1991b). The test substance was applied in concentrations of 3%, 10% and 30% on three consecutive days to the dorsum of both ears of 4 female CBA/Ca mice each. Five days later the animals received approx. 20 µCi of 3H-methly thymidine and were sacrificed 5 h later for measurement of radioactivity. No significant increase in isotope incorporation was detected after repeated application. The stimulation index, calculated by comparison of the CPM of the control and the treated animals, was 0.45 for the 3% application, 2.05 for 10% and 1.21 for the 30% application. According to the provider, the test substance contained up to 2% of an additional package which was not further defined. This might contribute to the result of this test. Therefore the result cannot be clearly assigned to the test substance and this study is not considered for classification.

In a fourth study, a Local Lymph Node Assay, was conducted analogously to the first one reported for the test substance, but with significant methodical deficiencies (Robinson, 1991c). The test substance was investigated at low concentrations of 3 and 10%. In addition, the test substance comprised additives which were not further specified. Therefore the results obtained do not allow the derivation of a dose dependency and are insufficient for assessment.

Migrated from Short description of key information:

Skin sensitisation: Non sensitiser in LLNA (Mouse).

Justification for selection of skin sensitisation endpoint:

Endpoint conclusion derived in LLNA in compliancce with OECD guideline 429, EU test standard B42 and USA EPA test guideline 870.2600

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification