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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 July 2003 to 28 October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
yes
Remarks:
animal room relative humidity range exceeded the preferred range
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
animal room relative humidity range exceeded the preferred range
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
animal room relative humidity range exceeded the preferred range
GLP compliance:
yes
Type of study:
Buehler test
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
ANIMAL DESCRIPTION, IDENTIFICATION AND HOUSING
- Young adult, Hartley-derived albino guinea pigs were received from Hilltop Lab Animals Inc., Scottdale, PA.
- Upon receipt, plastic ear tags displaying unique identification numbers were used to individually identify the animals.
- Cage cards displaying at least the study number, animal number and sex were affixed to each cage.
- The animals were housed individually in suspended stainless steel cages.
- All housing and care were based on the standards recommended by the Guide for the Care and Use of Laboratory Animals.

ENVIRONMENT
- The animal room temperature and relative humidity ranges were 64-73°F (18-23 °C) and 43-85 %, respectively.
- Environmental control equipment was monitored and adjusted as necessary to minimize fluctuations in the animal room environment. - Light timers were set to maintain a 12-hour light / l2-hour dark cycle and room ventilation was set to produce 10-15 air changes per hour.
- The room temperature and relative humidity were recorded a minimum of once daily.

FOOD
- PMI Certified Guinea Pig Chow #5026 (PMI Nutrition International) was provided ad libitum to the animals throughout the study.
- The lot number and expiration date of each batch of diet used during the study were recorded.
- The feed was analyzed and certified by the supplier for nutritional components and environmental contaminants.
- Dietary limitations for various environmental contaminants, including heavy metals, pesticides, polychlorinated biphenyls and total aflatoxin are set by the manufacturer. Within these limits, contaminants which may have been present were not expected to compromise the purpose of this study.

WATER
- Municipal tap water treated by reverse osmosis was available ad libitum throughout the study. The purified water was supplied by an automatic watering system.
- Monitoring of the drinking water for contaminants is conducted by Springborn and the records are available for inspection. Within generally accepted limits, contaminants which may have been present were not expected to compromise the purpose of this study. The water meets the standards specified under the EPA National Drinking Water Regulations (40 CFR Part 141).

ACCLIMATION
- Upon receipt, the animals were removed randomly from the shipping cartons, examined by qualified personnel, identified with plastic ear tags and then acclimated to the laboratory conditions for a minimum of five days.
- The animals were observed daily for overt physical or behavioral abnormalities, general health/moribundity and mortality.

ANIMAL SELECTION
- The animals chosen for study use were arbitrarily selected from healthy stock animals to avoid potential bias.
- All animals received a detailed pretest observation prior to dosing. Only healthy animals were chosen for study use.
- Females were nulliparous and nonpregnant.
- The male animals were approximately 7 weeks of age and weighed 304-423 g on the day prior to Induction 1 dosing.
- The female animals were approximately 8 weeks of age and weighed 340-389 g on the day prior to Induction 1 dosing.
Route:
epicutaneous, occlusive
Vehicle:
other: Mineral oil USP
Concentration / amount:
- Induction: 100 % test material
- Challenge: 50 % test material
- Challenge control: 50 % test material
- Rechallenge: 75 % test material
- Rechallenge control: 75 % test material
- Rechallenge control: 50 % test material
Route:
epicutaneous, occlusive
Vehicle:
other: Mineral oil USP
Concentration / amount:
- Induction: 100 % test material
- Challenge: 50 % test material
- Challenge control: 50 % test material
- Rechallenge: 75 % test material
- Rechallenge control: 75 % test material
- Rechallenge control: 50 % test material
No. of animals per dose:
- Induction: 10 males and 10 females
- Challenge: 10 males and 10 females
- Challenge control: 5 males and 5 females
- Rechallenge: 10 males and 10 females
- Rechallenge control (75 % test material) 5 males and 5 females
- Rechallenge control (50 % test material) 5 males and 5 females
Details on study design:
PRELIMINARY PROCEDURE
- On the day prior to each dose administration, the guinea pigs had the hair removed with a small animal clipper.
- Care was taken to avoid abrading the skin.

DOSING
A dose of 0.3 mL of the test article or 0.3 mL of HCA was placed on a 25 mm Hilltop chamber backed by adhesive tape (occlusive patch).
- The chambers were then applied to the clipped surface as quickly as possible.
- Following chamber application, the trunk of the animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chamber and the animal was returned to its cage.

INDUCTION
- On the day prior to the first induction dose administration (day -I), all test, HCA test, challenge/rechallenge/second rechallenge control and HCA control animals were weighed and the hair was removed from the left side of the test and HCA test animals.
- On the day following clipping (day 0), chambers were applied as shown in the attached table.
- The induction procedure was repeated on study day 7 and on study day 14 so that a total of three consecutive induction exposures were made to the test and HCA test animals.

CHALLENGE
- On the day prior to challenge dose administration, the test, HCA test, challenge control and HCA challenge control animals were weighed and the hair was removed from the right side of the animals.
- On the day following clipping (day 27), chambers were applied as shown in the attached table.

RECHALLENGE
- A rechallenge was conducted in order to substantiate and clarify the challenge results.
- On the day prior to rechallenge dose administration, all test and rechallenge control animals were weighed and the hair was then removed from
the right side of the animals.
- On the day following clipping (day 34), chambers were applied as shown in the attached table.

TEST ARTICLE REMOVAL
- Approximately six hours after chamber application, the binding materials were removed. The test sites were wiped with gauze moistened in deionised water, followed by dry gauze, to remove test article residue.
- The animals were then returned to their cages.

DERMAL OBSERVATIONS
- The test sites were graded for irritation at approximately 24 and 48 hours following chamber application (induction) or chamber removal (challenge and rechallenge) using the Dermal Grading System (attached) presented in Protocol Appendix B.

CLINICAL OBSERVATIONS
- Any unusual observations and mortality were recorded. The animals were observed for general healthlmortality twice daily, once in the morning and once in the afternoon.

BODY WEIGHTS
- Individual body weights were obtained for all sensitisation study animals on the day prior to the first induction (day - 1) and for the appropriate test and challenge control animals on the day prior to challenge dosing. The appropriate test and rechallenge control animals were weighed on the day prior to rechallenge dosing.

SCHEDULED EUTHANASIA
- Each sensitisation animal was euthanised by carbob dioxide inhalation following it final scoring interval.
- Gross necropsy examinations were not required for these animals.
Positive control substance(s):
yes
Remarks:
HCA
Positive control results:
- Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 were noted in 5/10 test animals at the 24-hour scoring interval.
- At the 48-hour scoring interval, dermal scores of 1 were noted in 3/10 test animals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 and ±. Group mean dermal scores were noted to be higher in the test animals as compared with the challenge control animals.
- Following challenge with 1% w/v HCA in acetone, dermal scores of 1 were noted in 2/10 test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be higher in the test animals as compared with the challenge control animals.

PROTOCOL DEVIATIONS

- The animal room relative humidity (43% to 85 %) exceeded the preferred range (30 % to 70 %) during the study. This occurrence was considered to have had no adverse effect on the outcome of the study.

TOPICAL RANGE-FINDING STUDY

- Individual topical range-finding data is presented in Appendix A (attached).

- The results of the range-finding study indicated that a test article concentration of 100% was considered appropriate for induction as the 100% concentration produced a mild to moderate dermal response.

- A concentration of 50% w/w in mineral oil USP, was considered appropriate for challenge as it produced minimal irritation which was considered to be the highest nonirritating concentration.

- Based on the challenge results, a rechallenge was conducted at the concentrations of 75% and 50% w/w in mineral oil USP, to further evaluate the sensitization response observed at challenge.

SENSITISATION DATA USING TEST MATERIAL

- Individual data are presented in Tables 1 to 5 (attached).

- Following challenge with 50% w/w test material in mineral oil USP, dermal scores of 1 were noted in 1/20 test animals at the 24-hour scoring interval. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be slightly higher in the test animals as compared with the challenge control animals.

- Following rechallenge with 75% w/w test material in mineral oil USP, dermal reactions in the test and rechallenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be slightly higher in the test animals as compared with the rechallenge control animals.

Following rechallenge with 50% w/w test material in mineral oil USP, dermal reactions in the test and rechallenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be slightly higher in the test animals as compared with the rechallenge control animals.

CLINICAL OBSERVATIONS AND BODY WEIGHTS

- Individual clinical observations are presented in Appendix D (attached).

- Individual body weight data are presented in Appendix E (attached).

- Animal No. G50631M (HCA control group) exhibited clinical observations of few faeces, decreased food consumption, shallow breathing, slow breathing, laboured breathing, activity decreased, dehydration (days 32 to 35), gasping (days 32 and 33), soft stools (day 33), wobbly gait, faeces small in size and impaired mobility; hindlimb (days 34 and 35). These findings were considered to be isolated since no other animals exhibited these findings.

- The sensitisation study animals gained weight during the test period and the remaining study animals generally appeared in good health.

Conclusions:
The test material is not considered to be a contact sensitiser in guinea pigs since the number of dermal responders was below the 15% requirement. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitisers (OECD 406).
Executive summary:

GUIDELINE

The dermal sensitization potential (delayed contact hypersensitivity) of the test item was evaluated in Hartley-derived albino guinea pigs when administered by multiple topical applications. The study was performed in accordance with US EPA, Health Effects Test Guidelines, OPPTS 870.2600, Skin Sensitization, March 2003, OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, Subsection 406, July 17, 1992 and the EEC Part B: Methods for the Determination of Toxicity, No. L 383 N131, B.6., December 29, 1992.

METHOD

Ten male and ten female guinea pigs were topically treated with undiluted test material, once per week for three consecutive weeks. Following a two week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naive) challenge control guinea pigs were topically treated with 50 % w/w test material in mineral oil USP. Challenge responses in the test animals were compared with those of the challenge control animals. Following a seven day rest period, a re-challenge was performed whereby the 20 test animals and 10 previously untreated (naive) rechallenge control guinea pigs were topically treated with 75 % and 50 % w/w test material in mineral oil USP. Rechallenge responses in the test animals were compared with those of the rechallenge control animals.

An α-hexylcinnamaldehyde (HCA) positive control group consisting of ten HCA test and ten HCA control guinea pigs was included in the study. The HCA test animals received 5 % w/v HCA in ethanol for induction and 2.5 % and 1.0 % w/v HCA in acetone for challenge.

RESULTS

The results of the range-finding study indicated that a test article concentration of 100 % was considered appropriate for induction as the 100 % concentration produced a mild to moderate dermal response. A concentration of 50 % w/w in mineral oil USP, was considered appropriate for challenge as it produced minimal irritation which was considered to be the highest nonirritating concentration. Based on the challenge results, a rechallenge was conducted at the concentrations of 75 % and 50 % w/w in mineral oil USP, to further evaluate the sensitization response observed at challenge.

Following challenge with 50% w/w test item in mineral oil USP, dermal scores of 1 were noted in 1/20 test animals at the 24-hour scoring interval. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be slightly higher in the test animals as compared with the challenge control animals.

Following rechallenge with 75% w/w test material in mineral oil USP, dermal reactions in the test and rechallenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be slightly higher in the test animals as compared with the rechallenge control animals.

Following rechallenge with 50% w/w test item in mineral oil USP, dermal reactions in the test and rechallenge control animals

0 to ±. Group mean dermal scores were noted to be slightly higher in the test animals as compared with the rechallenge control animals.

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 were noted in 5/10 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 were noted in 3/10 test animals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 and ±. Group mean dermal scores were noted to be higher in the test animals as compared with the challange control animals.

Following challenge with 1% w/v HCA in acetone, dermal scores of 1 were noted in 2/10 test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be higher in the test animals as compared with the challenge control animals.

Animal number G50631M (HCA control group) exhibited clinical observations of few faeces, decreased food consumption, shallow breathing, slow breathing, laboured breathing, activity decreased, dehydration (days 32 to 35), gasping (days 32 and 33), soft stools (day 33), wobbly gait, faeces small in size and impaired mobility; hindlimb (days 34 and 35). These findings were considered to be isolated since no other animals exhibited these findings.

The sensitization study animals gained weight during the test period and the remaining study animals generally appeared in good health.

CONCLUSION

Based on study results, the test material is not considered to be a contact sensitiser in guinea pigs since the number of dermal responders was below the OECD 15% requirement. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitisers.

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

GUIDELINE

The dermal sensitization potential (delayed contact hypersensitivity) of the test item was evaluated in Hartley-derived albino guinea pigs when administered by multiple topical applications. The study was performed in accordance with US EPA, Health Effects Test Guidelines, OPPTS 870.2600, Skin Sensitization, March 2003, OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, Subsection 406, July 17, 1992 and the EEC Part B: Methods for the Determination of Toxicity, No. L 383 N131, B.6., December 29, 1992.

METHOD

Ten male and ten female guinea pigs were topically treated with undiluted test material, once per week for three consecutive weeks. Following a two week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naive) challenge control guinea pigs were topically treated with 50 % w/w test material in mineral oil USP. Challenge responses in the test animals were compared with those of the challenge control animals. Following a seven day rest period, a re-challenge was performed whereby the 20 test animals and 10 previously untreated (naive) rechallenge control guinea pigs were topically treated with 75 % and 50 % w/w test material in mineral oil USP. Rechallenge responses in the test animals were compared with those of the rechallenge control animals.

Anα-hexylcinnamaldehyde (HCA) positive control group consisting of ten HCA test and ten HCA control guinea pigs was included in the study. The HCA test animals received 5 % w/v HCA in ethanol for induction and 2.5 % and 1.0 % w/v HCA in acetone for challenge.

RESULTS

The results of the range-finding study indicated that a test article concentration of 100 % was considered appropriate for induction as the 100 % concentration produced a mild to moderate dermal response. A concentration of 50 % w/w in mineral oil USP, was considered appropriate for challenge as it produced minimal irritation which was considered to be the highest nonirritating concentration. Based on the challenge results, a rechallenge was conducted at the concentrations of 75 % and 50 % w/w in mineral oil USP, to further evaluate the sensitization response observed at challenge.

Following challenge with 50% w/w test item in mineral oil USP, dermal scores of 1 were noted in 1/20 test animals at the 24-hour scoring interval. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be slightly higher in the test animals as compared with the challenge control animals.

Following rechallenge with 75% w/w test material in mineral oil USP, dermal reactions in the test and rechallenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be slightly higher in the test animals as compared with the rechallenge control animals.

Following rechallenge with 50% w/w test item in mineral oil USP, dermal reactions in the test and rechallenge control animals

0 to ±. Group mean dermal scores were noted to be slightly higher in the test animals as compared with the rechallenge control animals.

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 were noted in 5/10 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 were noted in 3/10 test animals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 and ±. Group mean dermal scores were noted to be higher in the test animals as compared with the challange control animals.

Following challenge with 1% w/v HCA in acetone, dermal scores of 1 were noted in 2/10 test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be higher in the test animals as compared with the challenge control animals.

Animal number G50631M (HCA control group) exhibited clinical observations of few faeces, decreased food consumption, shallow breathing, slow breathing, laboured breathing, activity decreased, dehydration (days 32 to 35), gasping (days 32 and 33), soft stools (day 33), wobbly gait, faeces small in size and impaired mobility; hindlimb (days 34 and 35). These findings were considered to be isolated since no other animals exhibited these findings.

The sensitization study animals gained weight during the test period and the remaining study animals generally appeared in good health.

CONCLUSION

Based on study results, the test material is not considered to be a contact sensitiser in guinea pigs since the number of dermal responders was below the OECD 15% requirement. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitisers.


Migrated from Short description of key information:
Based on study results, the test material is not considered to be a contact sensitiser in guinea pigs since the number of dermal responders was below the OECD 15% requirement. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitisers

Justification for selection of skin sensitisation endpoint:
GLP guideline study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for sensitisation according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on study results, the test material is not considered to be a contact sensitiser in guinea pigs since the number of dermal responders was below the 15% requirement of Regulation (EC) No 1272/2008.