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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Sep 2004 - 5 Oct 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to GLP and OECD Guideline READ ACROSS To address toxicological endpoints as part of the REACH registration of Lemonile (Target Substance) it is proposed to read-across to Hypo-Lem (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised in using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore read across is justified.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 01-230 (10).
-Chemical name: Octanenitrile, 3,7-dimethyl.
-Generic name: Hypo-Lem
- Physical state: Liquid
- Analytical purity: 98.3%
- Lot/batch No.: 295595
- Storage condition of test material: Room temperature protected from light
Specific details on test material used for the study:
- Name of test material (as cited in study report): 01-230 (10).
-Chemical name: Octanenitrile, 3,7-dimethyl.
-Generic name: Hypo-Lem
- Physical state: Liquid
- Analytical purity: 98.3%
- Lot/batch No.: 295595
- Storage condition of test material: Room temperature protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
CB6F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME 04609
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 19-26 g
- Housing: Animals were group housed (5 per cage)
- Diet: Animals had access to Certified Rodent Chow 7012C ad libitum
- Water: Tap water was available ad libitum
- Acclimation period: Study animals were acclimated to their housing for six days prior to their first day of dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 23.3 to 26.7 °C
- Humidity: 36-56 %
- Air changes (per hr):
- Photoperiod: 12 hrs light/12 hrs dark

Study design: in vivo (LLNA)

Vehicle:
other: Diethyl phthalate/ethanol in a ratio of 3:1
Concentration:
7.5%, 15% and 30% (test article)
35% (positive control)
No. of animals per dose:
5
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
On each day of dosing the test article was prepared at 7.5, 15 and 30% (w/v) in glass volumetric flask by dissolving the appropriate amount of test article in the vehicle. All preparations were vortexed to mix. The test article dosing solutions were clear colourless liquids.

On each day of doing, the positive control was prepared at 35% in glass tubes by adding the appropriate amount of HCA in the vehicle. Each preparation was vortexed to mix and was dosed as a clear yellow liquid. .



Mice were treated on the dorsal surface of both ears, once per day on Days 1, 2 and 3. Approximately 24 ± 2 hours between applications of test article was maintained. On day 6 the mice were injected i.v with 20µCi of 3H-thymidine in 250 µl of sterile saline. Five hours later the mice were euthanized by CO2 asphyxiation. Ear thickness measurements of each mouse were recorded and the draining auricular lymph nodes removed. At removal, the number of nodes collected per animal was recorded and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8°C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 ml of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in scintillation counte
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean DPM for each group was evaluated using a SYSTAT version 9.01, developed by SPSS, Inc. Increases in 3H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response is that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.

Body weight data and ear thickness measurements were also evaluated.

Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the DPM, bodyweight and ear thickness data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, a Dunnett’s test was used to determine the degree of significance from the control means.

Results and discussion

Positive control results:
The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 7.28. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control is considered a positive response. In addition, the response with the positive control in this study was also statistically significant (p<0.001) when compared to the vehicle control group.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Refer to Table 2 in results section
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Refer to Table 2 in results seciton

Any other information on results incl. tables

Table 1: Body weights

Group

Treatment

Dose

Body weights (g)

(mean ± sem)

Change in body weight

(g)

(mean ± sem)

Day 1

Day 6

1

Vehicle1

-

22.8 ± 0.8

22.8 ± 0.8

0.0 ± 0.0

2

04-230 (10)

7.5%

22.6 ± 1.0

23.0 ± 1.0

0.4 ± 0.2

3

04-230 (10)

15%

22.4 ± 1.2

23.4 ± 1.2

1.0 ± 0.0*

4

04-230 (10)

30%

22.6 ± 0.9

23.4 ± 1.2

0.8 ± 0.4

5

HCA

35%

22.0 ± 0.9

23.0 ± 0.7

1.0 ± 0.3*

1Diethyl phthalate in a ratio of 3:1

2Test/control ratio of 3.0 or greater represents a positive result

* Statistically significant difference when compared to the vehicle control group (Group 1) (p<0.05)

Table 2: Local Lymph Node Assay - Results

Group

Treatment

Dose

DPM

SI

Results

1

Vehicle1

-

186 ± 20

-

-

2

04-230 (10)

7.5%

251 ± 61

1.35

-

3

04-230 (10)

15%

444 ± 176

2.39

-

4

04-230 (10)

30%

226 ± 40

1.22

-

5

HCA

35%

1355 ± 107***

7.28

+

1Diethyl phthalate in a ratio of 3:1

2Test/control ratio of 3.0 or greater represents a positive result

*** Statistically significant difference when compared to the vehicle control group (Group 1) (p<0.001)

Table 3: Ear measurements

Group

Treatment

Dose

Ear measurement

(mm)

(mean ± sem)

1

Vehicle1

-

0.329 ± 0.006

2

04-230 (10)

7.5%

0.318 ± 0.004

3

04-230 (10)

15%

0.321 ± 0.004

4

04-230 (10)

30%

0.328 ± 0.006

5

HCA

35%

0.354 ± 0.008*

1Diethyl phthalate in a ratio of 3:1

2Test/control ratio of 3.0 or greater represents a positive result

* Statistically significant difference when compared to the vehicle control group (Group 1) (p<0.05)

All mice survived and appeared normal throughout the study. The application sites on the mice from the groups treated with the positive control appeared wet on days 2-4. There were no other findings.

 

At termination, the lymph nodes from the mice treated with the positive control were enlarged but appeared normal. The lymph nodes from the mice in the vehicle and all test treated animals were normal in size and appearance.

 

Mean bodyweights at day 1 and day 6 and mean changes in body weights were evaluated. There were no statistically significant differences observed in mean body weights between any of the treatment groups at Day 1 or Day 6. Statistically significant increases in the change in body weights were noted in the group treated with the test article at 15% and the group treated with the positive control when compared to the vehicle control However these differences were not biologically significant. Therefore, the test article did not cause any overt toxicity.

 

Exposure to 04-230 (10) at 7.5, 15 and 30% (w/v) resulted in stimulation indices of 1.35, 2.39 and 1.22 respectively. There were no statistically significant differences found at 7.5, 15 or 30% when the mean DPM for each treatment group was compared to the vehicle control group.

At termination both ears of each mouse were also measured. The only statistically significant difference in the ear measurements that was detected was an increase in the ear measurement for the possible control group when compared to the vehicle control group (P <0.05). However, this increase in ear thickness was only about 7.6% which is not biologically relevant.  

READ ACROSS

To address toxicological endpoints as part of the REACH registration of Lemonile (Target Substance) it is proposed to read-across to Hypo-Lem (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised in using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore read across is justified.

 See Section 13, document Read across justification_Hypo-Lem.

At termination both ears of each mouse were also measured. The only statistically significant difference in the ear measurements that was detected was an increase in the ear measurement for the possible control group when compared to the vehicle control group (P <0.05). However, this increase in ear thickness was only about 7.6% which is not biologically relevant.  

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
A test material is considered to have skin sensitizing activity, if at one or more concentrations it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index ≥ 3.0 is regarded as a positive result. Therefore based on the criteria of the study, treatment with 04-230 (10) did not result in a stimulation index of 3 or greater and hence it is not considered to have skin sensitizing activity.