1-Butanone, 3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-, rel-

Endpoint summary

Description of key information

Skin sensitisation:

- LLNA: Skin sensitizer (OECD 429, GLP, K, Rel.1)

- HRIPT: No evidence of sensitisation in humans when tested at 1% in DEP

Respiratory sensitisation: No data available.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 January 2004 to 10 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 429 and in compliance with GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Maximal dose tested was 40%, this did not affect the relevance of the test

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Physical state: solid at ambient temperature
- Storage condition of test material: ca. 4°C in the dark under nitrogen

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratory (Bar Harbor, ME, - USA)
- Age at study initiation: 6 to 7 weeks of age
- Weight at study initiation: 17.9-23.6 g
- Housing: individually in plastic shoebox-style cages at the North Carolina State University College of Veterinary Medicine.
- Diet (e.g. ad libitum): Purina Rodent Chow 5002 and City of Raleigh tap water ad libitum.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7-24.4
- Humidity (%): 17-60. Some humidity readings were outside the range specified in the OECD Guideline 429 (at least 30%). Minimum humidity readings in the animal room on 27, 28, 29 February and 1, 2 & 8 March were less than 30% (17% - 29%). This deviation is not expected to have any effect on data quality or integrity, as per a letter on file from the Director of Laboratory Animal Resources.
- Air changes (per hr): not mentioned in the study report
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: March 3, 2004 To: March 9, 2004

Vehicle:

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

Concentration:

1,, 5, 10 , 20 and 40 %

No. of animals per dose:

5 animals per dose (excepted for vehicle control: 8 animal per dose)

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A substance is considered a sensitizer if at least one concentration of the test material results in a
stimulation index (SI) greater than or equal to 3.

TREATMENT PREPARATION AND ADMINISTRATION: The test article, ST 48 C 03, was tested at 1%, 5%, 10%, 20% and 40%, final concentrations in acetone/olive oil (AOO; 4:1). The control articles included the vehicle (AOO) and a known sensitizer, isoeugenol, at 0.5 %, 1.0%, and 5.0%. The mice were restrained by hand, and 25 µL of test or control article was applied daily for three consecutive days to the dorsum of each ear using a calibrated Finnpipette. The animals were allowed to rest without dosing on Days 4 and 5. The mice were observed daily for signs of toxicity and mortality
On Day 6, individually numbered mice (labelled by tail markings) were injected in the lateral tail vein with 0.25 ml containing 2 µCi of I-125 labelled IuDR and 10.5 M FuDR (Sigma) in phosphate buffered saline (PBS). Approximately 5 hours later, the mice were euthanized by CO2 asphyxiation, and the auricular lymph nodes were excised. The nodes were dissociated using the frosted ends of glass slides. The cell suspension was washed with Hanks' balanced salt solution (HBSS) and then with PBS prior to being resuspended in 5% trichloroacetic acid (TCA, VWR) and refrigerated at approximately 4°C. Approximately 23 hours later, the cells were centrifuged and resuspended in fresh 5% TCA. The radioactivity was measured using a gamma counter (Packard Instruments).

Positive control substance(s):

other: isoeugenol (CAS: 97-54-1) at 0.5%, 1.0%, and 5.0%.

Statistics:

The natural log transformed DPM values for each compound were compared against vehicle by first performing a Bartlett's Chi-Square test for variance homogeneity. If this was found to be non-significant, a one-way analysis of variance was used using dose (concentration). If this was found to be significant, then a Dunnett's t test was performed using an alpha of 0.05. If the Barlett's Chi-Square was found to be significant, non-parametric analyses were performed. Specifically, a Kruskal-Wallis test was performed. If this was found to be significant, then a Jonckheere's-Terpera test was performed for dose-dependent trends.
A confirmatory analysis was performed against the known standard isoeugenol at three concentrations, 0.5%, 1%, and 5% using the above methods.

The data from the concentration tested was fit using a quadratic equation (a linear term and a square term of the concentration). If the quadratic term did not fit, a simple regression model was used. The concentration of chemical required to elicit an SI of 3 (EC-3) was determined from the appropriate regression equation.

Ear thickness measurements were checked for 10%-or-greater increases between Days 1 and 3. When applicable, the lowest concentration to elicit such an increase (minimal irritating concentration or MIC10) was determined. The MIC10 was then compared to the calculated EC-3. If the MIC10 was greater than the EC-3 (MIC10 > EC-3), confounding irritation is unlikely to have affected the LLNA. If the MIC10 was less than the EC-3 (MIC10 < EC-3), the concentration of the chemical that elicited the increase may have produced an LLNA stimulation index close to 3 because of the physiology surrounding the primary irritation reaction (Anderson et al., 2003).
All calculations were performed using Microsoft® Excel and SAS®, version 8. PROCs GLM, FREQ, NPARIWAY, and MEANS were utilized.

Positive control results:

An EC-3 of 0.88% was determined for the positive control isoeugenol in the current study using a linear regression method with a good fit. This is in agreement with the mean EC-3 value of isoeugenol of 1.2 +/- 0.6 % (Basketter & Cadby, Contact Dermatitis 2004 Jan;50(1):15-7). These data would classify isoeugenol as a strong sensitizer (potency value between 0.2 and 2%) and are consistent with previously reported results; this demonstrates the capability of the test laboratory to identify positive dermal sensitizers.

Key result

Parameter:

EC3

Value:

20.71

Key result

Parameter:

SI

Value:

5.3

Variability:

1.1

Test group / Remarks:

40%

Key result

Parameter:

SI

Value:

2.5

Variability:

0.3

Test group / Remarks:

20%

Key result

Parameter:

SI

Value:

1.5

Variability:

0.2

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1.6

Variability:

0.2

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

1

Variability:

0.2

Test group / Remarks:

1%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Mean DPM = 26.7, 41.3, 40.6, 65.3 and 140.4 at 1, 5, 10, 20 and 40%, respectively. DPM measurements for the first different concentrations of test material (1%) were similar to DPM measurement of vehicle. Then for higher concentration tested (5%, 10%, 20% and 40%), DPM increased in an exponential way: ~2.5 time relative DPM increase was measured using 20% of the test material and ~5 time increase was measured using 40% of the test material. For more details, see Remarks on results including tables and figures.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 1, 5, 10, 20 and 40% in tetrahydrofuran were 1, 1.6, 1.5, 2.5 and 5.3 respectively.

EC3 CALCULATION
The data from the concentration tested was fit using a quadratic equation (a linear term and a square term of the concentration). If the quadratic term did not fit, a simple regression model was used. The concentration of chemical required to elicit an SI of 3 (EC-3) was determined from the appropriate regression equation.
It was determined that the substance has an EC-3 of 20.71 %.

CLINICAL OBSERVATIONS:
None
None of the tested mice experienced 10%-Or-greater increases in ear thickness between Day 1 and Day 3, thus there was no irritation reaction to potentially affect the LLNA stimulation indices.

BODY WEIGHTS
Three mice, each in a different treatment group, lost minimal amonnts of weight between randomization and lymph node harvest (animals #11, 40, and 44 lost 0.3, 1.0, and 0.2 grams, respectively). Body weights at lymph node harvest mnged from 18.3 to 24.3 grams.

None of the mice assigned to this study experienced visible irritation or other adverse toxic effects after dosing. Three mice, each in a different treatment group, lost minimal amounts of weight between randomization and lymph node harvest (animals #11, 40, and 44 lost 0.3, 1.0, and 0.2 grams, respectively). On Day 1 of dosing, the tail of animal #7 was found to be raw, scaly, and inflamed. That mouse was removed from the study prior to dosing and replaced with animal #49.

 

Body weights at lymph node harvest ranged from 18.3 to 24.3 grams.

 

Individual and group DPM values and group SI values appear in Tables 1 and 2, respectively (See “Tables of results” in “background attached material”). All calculated DPM values were used in data calculations and statistical analyses.

 

A substance is considered a sensitizer if at least one concentration of the test material results in an SI value greater than or equal to 3. The test material had an SI value greater than 3 at the 40% concentration (SI ~ 5.3), as did isoeugenol at 5% (SI ~ 15.5). A one-sample, one-sided Student's t test found only the isoeugenol value statistically significant (p < 0.05). For ANOVA and non-parametric results, the isoeugenol model had excellent fit. Isoeugenol had a non-significant Bartlett's Chi-Square term; therefore, parametric results are reliable. Dunnett's test found only the 5.0% concentration of isoeugenol to be different from vehicle. The results of the KW and Jonckheere's tests were both significant, which confirms the parametric Dunnett's test results. The parametric ANOVA had excellent fit for the test material also. The Bartlett's Chi-Square term was non-significant; therefore, parametric resuIts are reliable.Dunnett's test found both the 20% and 40% concentrations of the test material to be different from vehicle. The results of the KW andJonckheere's tests were significant, confirming the parametric results and suggesting concentration differences and a dose response or trend.

The quadratic and linear models had excellent fit for isoeugenol, although the quadratic term was non significant.The EC-3 (linear model) was calculated to be 0.88% for isoeugenol. Both the quadratic and the linear model had excellent fit for the test material. The quadratic term was not significant; therefore the linear model is preferred. The calculated EC-3 for the test material, 20.71%, is within the acceptable range for the concentrations tested and is therefore considered reliable.

None of the tested mice experienced 10% or-greater increases in ear thickness between Day 1 and Day 3, thus there was no irritation reaction to potentially affect the LLNA stimulation indices. Ear measurements for individual mice follow in “Tables of results” in “background attached material".

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the conditions of testing, the substance is classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS, based on the calculated EC3 of 20.71 % (> 2%).

Executive summary:

In a dermal sensitization study performed according to the OECD test guideline No. 429 and in compliance with Good Laboratory Practice, the test material was tested in female CBA/J mice using the Local Lymph Node Assay. Mice were treated daily for three consecutive days by direct epicutaneous application of 25 µL of test or control article to the dorsum of each ear. The test material was tested at 1, 5, 10, 20, and 40 % final concentrations in acetone/olive oil. The control articles included the vehicle, acetone/olive oil (AOO), and a known sensitizer, isoeugenol. Isoeugenol was tested at 0.5, 1.0, and 5.0 % concentrations in AOO (4:1). The mice were observed daily. Three days after the final auricular application, the animals were injected intravenously with 125-I labelled Iododeoxyuridine to label proliferating cells. I-125-incorporation was quantified using a gamma counter.

A substance is considered a sensitizer if at least one concentration of the test material results in a stimulation index (SI) greater than or equal to 3. The test material had an SI value greater than 3 at the 40% concentration (SI = 5.3), as did isoeugenol at 5.0 % (SI = 15.5). Only isoeugenol at 5 % was found to be statistically significant (p < 0.05). None of the other tested concentrations yielded SI values greater than or equal to 3.

Mean ear thickness values, which were taken on Days 1 and 3, did not increase by 10 % or more at any tested concentration of either isoeugenol (0.5 %-5.0 %) or ST 48 C 03 (1 %-40 %).

Isoeugenol, with an EC-3 of 0.88 %, is classified as a strong sensitizer (potency value between 0.2 and 2%) (ECHA Chapter R.8, 2010). This is consistent with previously reported results.

The test material, with an EC-3 of 20.71 %, is classified as a moderate sensitizer (potency value above 2%).

Under the conditions of testing, the substance is classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS, based on the calculated EC3 of 20.71 % (> 2%).

Under the categorisation of chemicals according to skin sensitisation potency using the LLNA, the substance is a weak skin sensitiser (Potency Values from the Local Lymph Node Assay: Application to Classification, Labelling and Risk Assessment. ECETOC Document No. 46, December 2008).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A key study was identified (Burleson, 2004). In this dermal sensitization study performed according to the OECD test guideline No. 429 and in compliance with GLP, the test material was tested in female CBA/J mice using the Local Lymph Node Assay. The test material was tested at 1, 5, 10, 20, and 40 % final concentrations in acetone/olive oil. The control articles included the vehicle, acetone/olive oil (AOO), and a known sensitizer, isoeugenol.

The test material had an SI value greater than 3 at the 40% concentration (SI = 5.3), as did isoeugenol at 5.0 % (SI = 15.5). Only isoeugenol at 5 % was found to be statistically significant (p < 0.05). None of the other tested concentrations yielded SI values greater than or equal to 3.

Mean ear thickness values, which were taken on Days 1 and 3, did not increase by 10 % or more at any tested concentration of either isoeugenol (0.5 %-5.0 %) or ST 48 C 03 (1 %-40 %).

Isoeugenol, with an EC-3 of 0.88 %, is classified as a strong sensitizer (potency value between 0.2 and 2%) (ECHA Chapter R.8, 2010).

The test material, with an EC-3 of 20.71 %, is classified as a moderate sensitizer (potency value above 2%).

A Human Insult Repeated Patch Test (HRIPT) performed according to FDA regulations showed no evidence of sensitization in human in the conditions tested i.e. 1% in diethyl phtalate. (See §Sensitisation data (humans))

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Under the REACH regulation there is no legal standard information requirement in Annexes VII to X to perform any specific test for respiratory sensitisation. In addition, no validated or widely recognised in vitro or in vivo test methods specific to respiratory sensitisation are available yet. No human or animal data are available on the substance to address respiratory sensitisation. No alert for respiratory sensitisation were found by the OECD QSAR Toolbox.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

Based on the available data, the substance is classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS, based on the calculated EC3 of 20.71 % (> 2%).

No direct scientific data are available on the substance to address respiratory sensitisation.