Fatty acids, tall-oil, reaction products with boric acid (H3BO3) and diethanolamine

Endpoint summary

Description of key information

When assessed using the Local Lymph Node Assay, the test substance is considered to be a contact dermal sensitiser at concentrations of greater than or equal to 7 % (EC3 value) (OECD 429, EU Method B.42 and OPPTS 870.2600).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-02-29 to 2012-04-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

TEST ANIMALS
- Age at study initiation: 11 weeks
- Weight at study initiation: 19.2 - 25.4 g
- Housing: Plastic solid bottomed cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8-21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26°C
- Humidity (%): 45-53%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 2012-02-29 To: 2012-040-04

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary test: 80, 50, 25, 20, 10 and 5%
Main test: 10, 5 and 1%

No. of animals per dose:

5/dose level

Details on study design:

RANGE FINDING TESTS:
- Irritation: Scored for erythema pre-dose Days 1, 2, 3 and on Day 6. Ear-swelling measurements (duplicate) of each animal on Day 1 (pre-dose), Day 3 (ca. 48 hours after 1st dose) and Day 6 prior to scarifice.
- Lymph node proliferation response: Not measured

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Random assignment
- Criteria used to consider a positive response: Erythema and Oedema to modified Draize scoring method. Ear- swelling based on percentage change from control. Sensitisation respnse by B-scintillation (disintegrations per minute, dpm); mean dpm per test group /mean dpm of vehicle control group. SI > 3 = positive response.

TREATMENT PREPARATION AND ADMINISTRATION:
Dilutions of the test substance were prepared as w/w mixtures in acetone/olive oil (AOO 4:1 v/v). A single concentration of the positive control (25%w/w mixture of HCA in AOO) was prepared. All dosage preparations were freshly prepared on the day of administration.
Beginning on Day 1, 25µL of the appropriate test substance concentration, vehicle alone or the positive control substance was applied tothe dorsum of bpth ears of each mouse once per day for three consecutive days using a micropipette. During application, the material was spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette.
On Day 6 of the study ( 3 days after the final topical application) 250 µL of sterile phosphate buffered saline containing 20 µCi of 3-H-methyl thymidine was injected intravenously via the tail vein of each mouse.
Approximately 5 hours after the injection, the draining auricular lymph nodes from all animals were excised. The lymph nodes were pooled for each individual mouse. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gently massaging the lymph nodes between the frosted ends of two microscope slides ovaer a collection vessel. The slides were then briefly rinsed with PBS into the vessel. The vessel contents were centrifuged for approximately 10 minues at 1800 rpm twice and washed. After the second wash ca. 5 mL of 5% trichloracetic acid (TCA) in distilled water was added to the sediment and vortexed. The DNA was precipitated in the 5% TCA at 4.0 - 5.2°C overnight (ca. 18 hours).
After precipitation the tubes were centrifuged and the suprnatent discarded. The resulting precipitate was resuspended in 1 mL of 5% TCA and transferred to 10 mL scintillation fluid.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Significance judged at p<0.05. Treated groups and negative vehicle control group were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to control by Dunnett's t-test for multiple comparisons. Where variances are considered significantly different by Bartlett's test groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn's test). Outlier analysis was conducted using Grubbs (1969).

Parameter:

SI

Value:

0.95

Variability:

Group Mean DPM 1408.36

Test group / Remarks:

1% Test substance

Parameter:

SI

Value:

2.07

Variability:

Group Mean DPM 13077.36

Test group / Remarks:

5 % Test Substance

Parameter:

SI

Value:

3.96

Variability:

Group Mean DPM 5890.90

Test group / Remarks:

10 % Test Substance

Although nine mice lost or failed to gain body weight during the study, all animals appeared active and healthy throughout the study.

Group 1; Vehicle control: Very slight erythema noted at two test sites between Days 2 and/or 3

Group 2; Positive control: Very slight erythema noted at most sites between Days 2 and/or 6

Group 3; 1% test conc.: Very slight erythema noted at three sites between Days 2 and/or 6

Group 4; 5% test conc.: Very slight erythema noted at most sites between Days 2 and/or 6

Group 5; 10% test conc.: Very slight to well-defined erythema noted at all sites between Days 2 and/or 6

See Table 4 in attached document: LLNA_Durando 2012_Results Tables Treatment of mice with 1, 5 or 10% of the test material resulted in stimulation index values of 0.95, 2.07 and 3.96, respectively, relative to vehicle control mice. The substance was considered positive for a dermal sensitisation potential with a score > 3 at a test concentration at 10%. The positive control elicited a response of 4.66 relative to the vehicle control. The EC3 value was calculated to be 7%.

Interpretation of results:

sensitising

Conclusions:

The test substance is considered to be a contact dermal sensitiser at concentrations of greater than or equal to 7% (EC3 value)

Executive summary:

Test Guidance

OECD 429, EU Method B.42 and US EPA OPPTS 870.2600

Method and material

Three concentrations of the test substance in acetone/olive oil (AOO 4:1 v/v) or the vehicle alone were topically applied to twenty healthy mice (5/dose) for three consecutive days. Three days after the last application, the mice were given a 20 µCi IV injection of 3H-methyl thymidine. Five hours later, the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter. The results were presented as disintegrations per minutes per mouse (dpm/mouse). The ears of each animal were also evaluated for erythema and oedema prior to each application and again on Day 6, prior to the IV injection. A positive control group (5 animals) was maintained under the same environmental conditions and treated with 25% w/w mixture of alpha-hexylcinnamaldehyde (HCA) in AOO in the same manner as the test animals. Ear thickness (swelling) was taken on the preliminary, test and control animals on Day 1 (predose), Day 3 (ca 48 hours aftre the first dose), and Day 6 prior to sacrifice.

Results

Treatment of mice with 1, 5 or 10% of the test material resulted in stimulation index values of 0.95, 2.07 and 3.96, respectively, relative to vehicle control mice. The substance was considered positive for a dermal sensitisation potential with a score > 3 at a test concentration at 10%. The positive control elicited a response of 4.66 relative to the vehicle control. The EC3 value was calculated to be 7%.

Conclusions

The test substance is considered to be a contact dermal sensitiser at concentrations of greater than or equal to 7 % (EC3 value).

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 February 1983 to 28 April 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: Guinea Pig contact Dermal Irritation/sensitization - Modified Buehler Method

Deviations:

no

Principles of method if other than guideline:

Buehler, E.V., Delayed Contact Hypersensitivity in the Guinea Pig. Arch. Dermat.91; 171-175, 1965.

GLP compliance:

not specified

Type of study:

Buehler test

Justification for non-LLNA method:

The study was performed using the standard testing methodology at the time, prior to the adoption of the LLNA method.

Species:

guinea pig

Strain:

not specified

Sex:

male

Details on test animals and environmental conditions:

The animals were housed and maintained in accordance with standards set forth in the Guide for the Care and Use of Laboratory Animals (DHEW
Publication No. 80-23). The animals were not used in any previous study, and were acclimated to the laboratory for an appropriate time prior to dosing.

Husbandry Conditions:
Temperature - 65-75°F
Relative Humidity - 45-55%
Light - 12 hours light/dark cycle
Diet - Guinea pig Formula and tap water were provided ad libitum.
Caging - stainless steel with elevated wire mesh flooring
5 guinea pigs/cage by sex

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

1.0 % v/v

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

1.0 % v/v

No. of animals per dose:

10

Details on study design:

A group of ten male albino guinea pigs, weighing between 300 and 400 grams each, was employed in this study.
Irritation seen at several concentrations in preliminary topical screening studies indicated that the test should be performed with a 1.0% v/v dilution of the test material in mineral oil.
A 0.5 ml portion of diluted material was applied to the intact skin test sites on the guinea pigs.
A gauze patch was placed over the treated area and an impervious material was wrapped snugly around the trunks of the animals to hold the patch in
place.
After a 6 hour contact period the patch was removed and animals were allowed to rest for a minimum of one day. Following the rest period another application was applied to the same skin site using a fresh sample. This sequence was repeated three times weekly until a total of ten (10) applications had been made.
After the tenth application the animals were rested for a two week period before being challenged. The challenge application was applied to an area different than the induction site for 24 hours.
Twenty-four hours after each induction stage application and twenty-four and forty-eight hours after the challengeapplication, the sites were examined for irritation, if any, using the Draize method of scoring to grade reactions.

Challenge controls:

No data given

Positive control substance(s):

yes

Remarks:

1-chloro-2,4-dinitrobenzene

Positive control results:

Positive Control: 1-chloro-2,4-dinitrobenzene, as a 0.1% w/v suspension in physiological saline
The positive control material does not appear to be a primary skin irritant or fatiguing agent. It does appear to be a sensitizer in the albino guinea pig.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1.0% v/v in mineral oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no data given

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1.0% v/v in mineral oil. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no data given.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1.0% v/v in mineral oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no data given

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1.0% v/v in mineral oil. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no data given.

See attached - Table 1 Results

Interpretation of results:

not sensitising

Conclusions:

The material does not appear to be a primary skin irritant, fatiguing agent or sensitizer in the albino guinea pig.

Executive summary:

Test Guidance

Buehler, E.V., Delayed Contact Hypersensitivity in the Guinea Pig. Arch. Dermat.91; 171-175, 1965.

Method and Materials

The test material was applied as a 1.0 % v/v dilution in mineral oil A group of ten male albino guinea pigs, weighing between 300 and 400 grams each, was employed in this study. Irritation seen at several concentrations in preliminary topical screening studies indicated that the test should be performed with a 1.0% v/v dilution of the test material in mineral oil. A 0.5 ml portion of diluted material was applied to the intact skin test sites on the guinea pigs. A gauze patch was placed over the treated area and an impervious material was wrapped snugly around the trunks of the animals to hold the patch in place. After a 6 hour contact period the patch was removed and animals were allowed to rest for a minimum of one day. Following the rest period another application was applied to the same skin site using a fresh sample. This sequence was repeated three times weekly until a total of ten (10) applications had been made. After the tenth application the animals were rested for a two week period before being challenged. The challenge application was applied to an area different than the induction site for 24 hours. Twenty-four hours after each induction stage application and twenty-four and forty-eight hours after the challenge application, the sites were examined for irritation, if any, using the Draize method of scoring to grade reactions.

Results

0/10 test animals showed a sensitisation reaction under the conditions of the study

Conclusion

The material does not appear to be a primary skin irritant, fatiguing agent or sensitizer in the albino guinea pig.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10-07-2012 to 23-08-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
The male range-finding animals were approximately 5 weeks of age on the day prior to dosing with body weights of 325 to 343 grams. The female range-finding animals were approximately 6 weeks of age on the day prior to dosing with body weights of 336 to 351 grams.
The male second range-finding animals were approximately 9 weeks of age on the day prior to dosing with body weights of 414 to 456 grams. The female second range-finding animals were approximately 10 weeks of age on the day prior to dosing with body weights of 461 to 463 grams.
The male main phase animals were approximately 6 weeks of age on the day prior to Induction 1 dosing with body weights ranging from 348 to 447 grams. The female main phase animals were approximately 7 weeks of age on the day prior to Induction 1 dosing with body weights ranging from 337 to 395 grams.
- Housing: solid bottom cages containing direct bedding material. As an alternative, guinea pigs were individually housed in solid bottom cages containing direct bedding material.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20ºC to 26ºC
- Humidity (%): 50% ± 20%
- Air changes (per hr): 10 or more air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 10 Jul 2012 To: 31 Aug 2012

Route:

epicutaneous, occlusive

Vehicle:

other: white mineral oil, USP

Concentration / amount:

Dose range finding:
1st: 100, 75, 50 and 25%
2nd: 50, 30, 20 10%
Main Phase:
Induction: 100%
Challenge: 40%

Route:

epicutaneous, occlusive

Vehicle:

other: white mineral oil, USP

Concentration / amount:

Dose range finding:
1st: 100, 75, 50 and 25%
2nd: 50, 30, 20 10%
Main Phase:
Induction: 100%
Challenge: 40%

No. of animals per dose:

Range finder: 2/sex/dose
10/sex challenge: 5/sex control = 20 challenge: 10 control

Details on study design:

RANGE FINDING TESTS:
Topical dosing
0.3 mL of one of 4 concentrations was applied to the clipped area of an animal. Chambers were attached with elastic wrap. The chamers were removed after 6 hours and test sites wiped 2x with gauze moistened in mineral oil, then dry gauze, gauze moistened in distille water and finally dry gauze again.
Sites were checked after 24 and 48 hours.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 hours
- Test groups: 10 male/10 female
- Control group: No
- Site: left side of test animals
- Frequency of applications: 1 per induction
- Duration: 14 days
- Concentrations: 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Challenge started on day 28
- Exposure period: 6 hours
- Test groups: 10 male/10 female
- Control group: 5 male/5 female
- Site: right side
- Concentrations: 40%
- Evaluation (hr after challenge): 24 and 48 hours

Challenge controls:

Control animals not dosed suring induction phase.

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

40%

No. with + reactions:

17

Total no. in group:

20

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 40%. No with. + reactions: 17.0. Total no. in groups: 20.0. Clinical observations: None.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

40%

No. with + reactions:

16

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 40%. No with. + reactions: 16.0. Total no. in groups: 20.0. Clinical observations: none.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

40%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 40%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results table in attached document.

Range-Finding Phase

Exposure to test material at concentrations of 25%, 50%, 75%, and 100% resulted in dermal scores of 0 or ±. Therefore, induction was determined to be acceptable at 100% (as received). 

A second range-finding study conducted at concentrations of 10%, 20%, 30%, and 50% to determine an appropriate challenge level resulted in dermal scores of 0. Therefore, challenge was determined to be acceptable at 40% (w/w) in mineral oil, based on the results of the 2 range-finding studies.

Main Phase

During the induction, dermal scores of 0, ± (slight patchy erythema), 1 (slight, confluent or moderate patchy erythema), and 2 (moderate, confluent erythema) were noted for the test animals. Additional observations included edema scores of 1, superficial lightening, blanching, eschar, and desquamation.

Following challenge with 40% test material (w/w) in mineral oil, dermal scores of ± or 1 were noted in 17/20 test animals at the 24-hour scoring interval and in 16/20 test animals at the 48-hour scoring interval. No dermal irritation was observed in the control animals. Group mean dermal scores were higher in the test animals compared to the control animals.

Bodyweight

No test material-related effects on body weight were observed in the test animals during the study. Weight gain in the animals throughout the study interval was indicative of good health in the test and control animals.

Interpretation of results:

sensitising

Conclusions:

Based on the results and evaluation criteria of this study, the test material is considered a contact sensitizer under the conditions tested.

Executive summary:

Guideline

OECD Guideline 406 and EPA Health Effects Test Guideline OPPTS 870.2600.

Method and material

The dermal sensitization potential of test material was evaluated in Hartley-derived albino guinea pigs. Ten male and 10 female guinea pigs were topically treated with 100% (as received) test material once per week, for 3 consecutive weeks. Following a 2-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naïve) challenge control guinea pigs were topically treated with 40% test material (w/w) in mineral oil. Challenge responses in the test animals were compared to those of the challenge control animals. 

Range-finding Phase

Exposure to test material at concentrations of 25%, 50%, 75%, and 100% resulted in dermal scores of 0 or ±. Therefore, induction was determined to be acceptable at 100% (as received). 

A second range-finding study conducted at concentrations of 10%, 20%, 30%, and 50% to determine an appropriate challenge level resulted in dermal scores of 0. Therefore, challenge was determined to be acceptable at 40% (w/w) in mineral oil, based on the results of the 2 range-finding studies.

Results

Following challenge with 40% test material (w/w) in mineral oil, dermal scores of ± or 1 were noted in 17/20 test animals at the 24-hour scoring interval and in 16/20 test animals at the 48-hour scoring interval. No dermal irritation was observed in the control animals. Group mean dermal scores were higher in the test animals compared to the control animals.

Conclusions

Based on the results and evaluation criteria of this study, test material is considered a contact sensitizer under the conditions tested. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Key study

The key study was a Local Lymph Node Assay conducted in accordance with OECD 429, EU Method B.42 and US EPA OPPTS 870.2600. Three concentrations of the test substance in acetone/olive oil (AOO 4:1 v/v) or the vehicle alone were topically applied to twenty healthy mice (5/dose) for three consecutive days. Three days after the last application, the mice were given a 20 µCi IV injection of 3H-methyl thymidine. Five hours later, the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter.

The results were presented as disintegrations per minutes per mouse (dpm/mouse). The ears of each animal were also evaluated for erythema and oedema prior to each application and again on Day 6, prior to the IV injection. A positive control group (5 animals) was maintained under the same environmental conditions and treated with 25% w/w mixture of alpha-hexylcinnamaldehyde (HCA) in AOO in the same manner as the test animals. Ear thickness (swelling) was taken on the preliminary, test and control animals on Day 1 (predose), Day 3 (approximately 48 hours after the first dose), and Day 6 prior to sacrifice.

Treatment of mice with 1, 5 or 10% of the test material resulted in stimulation index values of 0.95, 2.07 and 3.96, respectively, relative to vehicle control mice. The substance was considered positive for a dermal sensitisation potential with a score > 3 at a test concentration at 10 %. The positive control elicited a response of 4.66 relative to the vehicle control. The EC3 value was calculated to be 7 %. The test substance is considered to be a contact dermal sensitiser at concentrations of greater than or equal to 7 % (EC3 value).

A local dermal DNEL can be derived from the results of the LLNA: The general approach for assessing and controlling risks and setting DNELs for skin sensitizers is outlined in the ECHA Guidance Chapter R.8. The first step should always be a qualitative approach by using potency categorization to define risk management measures (RMMs) and operational conditions (OCs). Data derived from the LLNA can serve for the potency categorization and is based on EC3 value. The second step is setting a DNEL to judge the remaining/residual likelihood of risks after the appropriate RMMs and OCs are implemented. It is suggested that the LLNA EC3 value correlates relatively well with the NOEL from human sensitization tests designed to confirm lack of induction (Gerberick et al., 2001; Griem et al., 2003; Basketter et al., 2005). Therefore, a threshold for skin sensitization can be defined as EC3 concentration (%), which is converted into dose per skin area (in µg/cm2):

 

EC3 [%] x ρ x 250 [µg/cm²/%] = EC3 [µg/cm²] = NOEL where ρ is density of the liquid, 250 factor is calculated by converting 25 µL (applied to the mouse ear in a standard LLNA protocol) into µg/cm².

 

LLNA descriptor:

EC3 [%] x ρ x 250 [µg/cm²/%] = 7 x 0.99 x 250 = 1732.5 [µg/cm²]

 

Dermal sensitization DNEL:

In order to calculate a DNEL for sensitization, sensitization uncertainty (assessment) factors must be applied to the NOEL (in this case the EC3 value).

¾    Interspecies variation = 1.

Skin sensitization is considered as local immunological effect. Mechanistically, the biological sequelae that take place for the immune system to mount a response are the same across mammalian species, so, toxicokinetic and toxicodynamic Assessment Factors are not applicable (Api, 2006; Basketter, 2000)

¾    Intraspecies (human-human inter-individual variation) = 5

¾    Dose response = 3.

Linear relationship was observed in the study (r2= 0.9932), and the test material is shown to be moderate skin sensitizer.

 

The final assessment factor is 15.

DNEL = 1732.5 µg/cm² ÷15 = 115.5 [µg/cm²]

Supporting study 1

The first supporting study was conducted in accordance with Buehler, E.V., Delayed Contact Hypersensitivity in the Guinea Pig. Arch. Dermat.91; 171-175, 1965. The test material was applied as a 1.0 % v/v dilution in mineral oil. A group of ten male albino guinea pigs, weighing between 300 and 400 grams each, was employed in this study. Irritation seen at several concentrations in preliminary topical screening studies indicated that the test should be performed with a 1.0% v/v dilution of the test material in mineral oil. A 0.5 ml portion of diluted material was applied to the intact skin test sites on the guinea pigs. A gauze patch was placed over the treated area and an impervious material was wrapped snugly around the trunks of the animals to hold the patch in place. After a 6 hour contact period the patch was removed and animals were allowed to rest for a minimum of one day. Following the rest period another application was applied to the same skin site using a fresh sample. This sequence was repeated three times weekly until a total of ten (10) applications had been made. After the tenth application the animals were rested for a two week period before being challenged. The challenge application was applied to an area different than the induction site for 24 hours. Twenty-four hours after each induction stage application and twenty-four and forty-eight hours after the challenge application, the sites were examined for irritation, if any, using the Draize method of scoring to grade reactions. No test animals (0/10) showed a sensitisation reaction under the conditions of the study and it was concluded that the material does not appear to be a primary skin irritant, fatiguing agent or sensitizer in the albino guinea pig.

Supporting study 2

The second supporting study was conducted in accordance with OECD Guideline 406 and EPA Health Effects Test Guideline OPPTS 870.2600. The dermal sensitization potential of test material was evaluated in Hartley-derived albino guinea pigs. Ten male and 10 female guinea pigs were topically treated with 100% (as received) test material once per week, for 3 consecutive weeks. Following a 2-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naïve) challenge control guinea pigs were topically treated with 40% test material (w/w) in mineral oil. Challenge responses in the test animals were compared to those of the challenge control animals. 

Exposure to test material at concentrations of 25 %, 50 %, 75 %, and 100 % resulted in dermal scores of 0 or ±. Therefore, induction was determined to be acceptable at 100% (as received). 

A second range-finding study conducted at concentrations of 10%, 20%, 30%, and 50% to determine an appropriate challenge level resulted in dermal scores of 0. Therefore, challenge was determined to be acceptable at 40% (w/w) in mineral oil, based on the results of the 2 range-finding studies.

Following challenge with 40 % test material (w/w) in mineral oil, dermal scores of ± or 1 were noted in 17/20 test animals at the 24-hour scoring interval and in 16/20 test animals at the 48-hour scoring interval. No dermal irritation was observed in the control animals. Group mean dermal scores were higher in the test animals compared to the control animals. Based on the results and evaluation criteria of this study, test material is considered a contact sensitizer under the conditions tested. 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with EU CLP Regulation (EC) No. 1272/2008, classification of this substance is required for skin sensitisation: Category 1B, H317 may cause an allergic skin reaction.