3-(chloromethyl)heptane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-(chloromethyl)heptane

EC Number:

204-594-4

EC Name:

3-(chloromethyl)heptane

Cas Number:

123-04-6

Molecular formula:

C8H17Cl

IUPAC Name:

3-(chloromethyl)heptane

Details on test material:

- Name of test material: 2-Ethylhexylchloride
- CAS-No.: 123-04-6
- Appearance: Colourless, clear liquid
- Purity: 99.4 corr. area-% determined by Capillary GC; dose calculation not adjusted to purity
- Batch No.: B023 000STD77L0
- Expiration Date: June 13, 2012
- Homogeneity: The test substance appeared to be homogeneous.
- Storage: At room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaCrl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Manston Road, Margate, Kent CT9 4LT, United Kingdom
- Age at study initiation: 8 – 9 weeks
- Weight at study initiation: 18 ± 1.2 g (mean ± SD)
- Housing: group housing (5 animals per group) in Makrolon Type II (pre-test)/ III (main study) cages, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water: Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Relative Humidity (%): 45 - 108
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. - 6.00 a.m. / 6.00 a.m. - 6.00 p.m.)

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25, 50% (w/w), and 100% (undiluted test item)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was the undiluted test item (100%). Test item solution at different concentrations was prepared using acetone:olive oil (4+1 v/v) as vehicle. Vortexing was used to prepare the test item formulations.
- Irritation: To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, a pre-test was performed in two animals. Two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50% (w/w) and 100% (undiluted test item) once daily each on three consecutive days. At the tested concentrations the animals did not show any signs of systemic toxicity. Only on day 4 and 5 of the pre-test, an erythema of the ear skin (Score 1 at 50% test item concentration and Score 2 using 100% of the undiluted test item) was observed. Furthermore, slightly flaky ear skin was seen on day 5 using 100% test item concentration. Thus, the test item in the main study was assayed at 25, 50% (w/w) and 100%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation in the pre-experiment.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index. Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and acetone:olive oil (4+1 v/v) was added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 25, 50% (w/w), and 100% (undiluted test item) in acetone:olive oil (4+1 v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) were treated with an equivalent volume of the relevant vehicle alone.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. The EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c, where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The periodic positive control experiment was performed with α-hexyl cinnamaldehyde dissolved in acetone:olive oil (4:1 v/v) (compound listed in OECD 429 Guideline) using CBA/CaCrl mice in August 2011. An EC3 value of 12.1% (w/v) was calculated.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Stimulation Indices (S.I.) of 2.12, 4.54, and 6.98 were determined with the test item at concentrations of 25, 50, and 100% (w/w) in acetone:olive oil (4+1 v/v), respectively, and an EC3 value of 34.1 % (w/w) was calculated.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The DPM values showed a statistically significant and biologically relevant increase in all dose groups in comparison to the vehicle control group and furthermore, a clear dose dependent increase in DPM value was observed.

Any other information on results incl. tables

Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation	SD	S.I.
	Mean DPM per animal (2 lymph nodes)^a)
Vehicle Control (acetone:olive oil (4+1 v/v))	397.8	128.2	1.00
25% 2-Ethylhexylchloride	843.6*	440.0	2.12
50% 2-Ethylhexylchloride	1805.2*	192.3	4.54
100% 2-Ethylhexylchloride	2778.2*	215.5	6.98

a)   Mean DPM/animal was determined by dividing the sum of the measured values from lymph
      nodes of all animals within a group by the number of animals in that group (5 animals)

*    Statistically significant increase vs. control group (p<0.05)

Calculation of the EC3 value:

	Test item concentration %	S.I.
Test Group 2	25 (a)	2.12 (b)
Test Group 3	50 (c)	4.54 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 34.1%(w/w)

EC3 = Estimated concentration for a S.I. of 3.

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

 

 

Viability / Mortality: No deaths occurred during the study period.

 

Clinical Signs: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

 

Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with ³HTdR, was within the range commonly recorded for animals of this strain and age.

 

Lymph Node Weights and Cell Counts: The measured lymph node weights and – cell counts of all animals treated were recorded after sacrifice. A statistically significant and biologically relevant increase in lymph node weight and cell count was observed in all treated groups in comparison to the vehicle control group (p<0.05). In both parameters, a clear dose response was seen. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. The cutoff-value was exceeded in all test item treated groups.

 

Ear Weights: The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in ear weights was not observed in any test item treated group in comparison to the vehicle control group.

For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response. None of the indices obtained in any group exceeded this threshold.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The test item 2-Ethylhexylchloride was found to be a skin sensitiser under the test conditions of this study.

Executive summary:

The study was performed according to OECD guideline 429 in compliance with GLP.

 

In this study the test item 2-Ethylhexylchloride was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in CBA/CaCrl mice. Test item solutions at 25, 50, and 100% (w/w) were prepared in the vehicle acetone:olive oil (4+1 v/v).

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

Stimulation Indices (S.I.) of 2.12, 4.54, and 6.98 were determined with the test item at concentrations of 25, 50, and 100% (w/w) in acetone:olive oil (4+1 v/v), respectively, and an EC3 value of 34.1% (w/w) was calculated. The DPM values showed a statistically significant and biologically relevant increase in all dose groups in comparison to the vehicle control group and furthermore, a clear dose dependent increase in DPM value was observed. This finding was confirmed by the lymph node weight and lymph node cell count, where a statistically significant (p<0.05) and biologically relevant increase was observed in all dose groups in comparison to the vehicle control group. A dose response was also present. The cutoff-value of 1.55 for the lymph node cell count index reported for BALB/c mice for a positive response was exceeded in all test item treated groups.

Conclusion: The test item 2-Ethylhexylchloride was found to be a skin sensitiser under the test conditions of this study.