Registration Dossier

Toxicological information

Respiratory sensitisation

Currently viewing:

Administrative data

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Remarks:
The reliability of the study cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cut-off value). No evaluation of irritation to the respiratory tract was made thus it cannot be determined if the level of irritation was excessive or not.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2007

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In respiratory lymph node assay, mice were exposed with test material via the respiratory route on three consecutive days. The immune response was determined by measuring cell proliferation and cytokine responses in the lymph nodes draining the respiratory tract. In this model, the most pronounced effects were found in the mandibular lymph nodes, which drain the nasopharynx.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Isoeugenol
- Source: Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands.
- Analytical purity: >99 %
- Isomers composition: not reported

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RIVM institute’s own breeding colony
- Age at study initiation: 6-8 weeks
- Housing: Animals were housed in macrolon cages under conventional conditions.
- Diet: Hope Farms chow pellets (Woerden, The Netherlands), ad libitum
- Water: Water, ad libitum

Test system

Route of induction exposure:
inhalation
Remarks:
vaporized or nebulized
Vehicle:
other: vaporized (no vehicle) or nebulized (acetone)
Concentration:
Vaporization using maximum vapour pressure: 11 ppm
Nebulization in acetone: 300 ppm
No. of animals per dose:
6 males
Details on study design:
EXPOSURE
- No. of exposures: 3
- Exposure period: 3 days
- Test groups: Mice were exposed nose-only to test material for 45, 90, 180 or 360 minutes per day on day 0, 1 and 2.
- Control group: Controls were exposed to the vehicle for 360 minutes per day on day 0, 1, and 2.
- Frequency of applications: Three
- Duration: 5 days
- Concentrations: Vaporization using maximum vapour pressure: 11 ppm; Nebulization in acetone: 300 ppm
- Dermal control was included. Mice were topically exposed to 10% test material in acetone: olive oil 4:1 (AOO) on the dorsum of both ears (25 μL/ear) on day 0, 1 and 2. Control mice received the same treatment with the vehicle (AOO).
Challenge controls:
Not applicable
Positive control substance(s):
not specified
Negative control substance(s):
not specified

Results and discussion

Results:
Effects of exposure to test material via maximum vapour pressure:
- Exposure to test material (11 ppm) increased cell number and cell proliferation in the mandibular LNs. The increase in cell proliferation did not show a time-dependent effect and the response was highly variable. None of the observed effects were statistically significant.
- Exposure to test material did not increase proliferation in the auricular LNs. However, the cell number in the control group was higher than normally observed. Therefore, the calculated SIs are below 1.
- Dermal application of test material (10%) resulted in a SI of the auricular LNs of 29.3.

Effects of exposure to aerosols of test material
- Exposure to aerosols of test material (300 ppm) resulted in toxic effects in the mice that were exposed for 360 min/day. After two days of exposure one mouse died and the other mice displayed several signs of distress. These mice were not exposed to test material on the third day. Effects of the two days exposure to test material were assessed on day 5. On the third day two mice died that were exposed for 180 minutes/day for 3 days. The other mice in this group appeared normal.
- Exposure to test material aerosols resulted in a significant increase of cell number and cell proliferation in the mandibular LNs. This increase was time-dependent, with the exception of the group that was exposed for 360 minutes/day. This group, however, was exposed for two days only. Effects of test material on cell number and cell proliferation were statistically significant for all exposure groups.
- Test material exposure for 90 minutes/day or longer increased proliferation in the auricular LNs. The mean SI in the auricular LNs was a factor 3-4 higher than in the mandibular LNs, but the variance was very high. Dermal exposure to 10% test material resulted in a SI of 18.8.
Positive control results:
None
Negative control results:
None

Any other information on results incl. tables

Effects of exposure to test material via maximum vapour pressure

 

Table 7.4.2/2: Effects of test material on mandibular LNs: cell number, cell proliferation and Sis

 

Group

Cell number

Proliferation

Stimulation index

Control

2.81 ± 1.44

1232 ± 596

1.0 ± 0.48

45 min/day

4.57 ± 1.49

3150 ± 1004

2.56 ± 0.81

90 min/day

5.69 ± 2.79

4383 ± 3530

3.56 ± 2.86

180 min/day

5.37 ± 1.20

2952 ± 711

2.40 ± 0.58

360 min/day

7.03 ± 2.19

4815 ± 1575

3.58 ± 1.40

 

Table 7.4.2/3: Effects of test material on auricular LNs: cell number, cell proliferation and Sis

 

Group

Cell number

Proliferation

Stimulation index

Inhalatory exposure

 

 

 

Control

6.46 ± 1.82

4406 ± 1766

1.0 ± 0.4

45 min/day

3.70 ± 1.34

1531 ± 414

0.35 ± 0.09

90 min/day

3.95 ± 0.98

1640 ± 477

0.37 ± 0.011

180 min/day

4.64 ± 1.35

2068 ± 919

0.47 ± 0.21

360 min/day

4.41 ± 1.63

1955 ± 584

0.43 ± 0.13

Dermal exposure

 

 

 

Control

3.92 ± 2.00

1278 ± 221

1.0 ± 0.17

10% test material

23.7 ± 6.82

37477 ± 13144

29.3 ± 16.7

 

Results are shown as mean ± SEM (n=6 per group). Cell number is expressed as 106 cells, proliferation is expressed as cpm per mouse. SIs are calculated by dividing the [3H]-thymidine incorporation of the experimental group with the mean [3H]-thymidine incorporation of the control group.

 

Effects of exposure to aerosols of test material

 

Table 7.4.2/4: Effects of test material on mandibular LNs: cell number, cell proliferation and Sis

 

Group

Cell number

Proliferation

Stimulation index

Control

2.34 ± 0.57

1070 ± 325

1.0 ± 0.30

45 min/day

5.18 ± 0.77**

4329 ± 688*

4.04 ± 0.64*

90 min/day

6.06 ± 1.41***

5486 ± 2515**

5.13 ± 2.35**

180 min/daya

5.14 ± 1.32**

6555 ± 2423***

6.13 ± 2.26***

360 min/dayb

5.54 ± 1.19**

4864 ± 1532*

4.54 ± 1.43*

 

Table 7.4.2/5: Effects of test material on auricular LNs: cell number, cell proliferation and Sis

 

Group

Cell number

Proliferation

Stimulation index

Inhalatory exposure

 

 

 

Control

3.38 ± 0.50

1493 ± 229

1.0 ± 0.15

45 min/day

4.74 ± 1.06

2528 ± 863

1.69 ± 0.58

90 min/day

10.7 ± 7.18

29549 ± 39947

19.79 ± 26.76

180 min/daya

10.7 ± 4.06

33648 ± 38004

22.54 ± 25.46

360 min/dayb

8.1 ± 3.12

15581 ± 15115

10.44 ± 10.12

Dermal exposure

 

 

 

Control

3.31 ±0.87

1698 ± 472

1.0 ± 0.28

10% test material

20.0 ±5.01

31941 ± 21910

18.8 ± 8.3

 

Results are shown as mean ± SEM (n=6 per group).a n=4; exposure for 3 days;b n=5, exposure for 2 days. Cell number is expressed as 106 cells, proliferation is expressed as cpm per mouse. SIs are calculated by dividing the [3H]-thymidine incorporation of the experimental group with the mean [3H]-thymidine incorporation of the control group. Statistically significant differences were assessed with a one-way ANOVA with a Bonferonni’s post hoc test. Asterisks depict significant differences from the control group: * p<0.05, ** p<0.01, *** p<0.001.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The authors considered that the study provided evidence of respiratory sensitisation. The reliability of the study cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cutoff value). In addition, no evaluation of irritation to the respiratory tract was made, although Isoeugenol is classified as skin and eye irritant and irritation has been observed in sub-acute inhalation toxicity studies. In the skin LLNA it is required to use dose levels that do not induce excessive levels of irritation. In this study it cannot be determined if the level of irritation was excessive or not, but in some case animals died and showed signs of respiratory distress. Furthermore, there is a potential weakness in the model in that the mandibular lymph node drain the mouth and nasophanrynx but not the tracheal and lung tissues, therefore the relevance to respiratory sensitisation is not proven.
Executive summary:

In a respiratory lymph node assay, groups of male Balb/c mice were exposed with isoeugenol (racemic mixture) through inhalation, either via vaporization using maximum vapour pressure (11 ppm) or via nebulization in acetone (300 ppm). Mice were exposed nose-only to test material for 45, 90, 180 or 360 minutes per day on day 0, 1 and 2. Controls were exposed to the vehicle for 360 minutes per day on day 0, 1 and 2. In this experiment a dermal control was included. Mice were topically exposed to 10% test material in acetone: olive oil 4:1 (AOO) on the dorsum of both ears (25 μL/ear) on day 0, 1 and 2. Control mice received the same treatment with the vehicle (AOO). At day 5, animals were killed and the auricular and mandibular lymph nodes were excised. The immune response was determined by measuring cell proliferation and cytokine responses in the lymph nodes draining the respiratory tract. 

 

Effects of exposure to test material via maximum vapour pressure: Exposure to test material (11 ppm) increased cell number and cell proliferation in the mandibular LNs. The increase in cell proliferation did not show a time-dependent effect and the response was highly variable. None of the observed effects were statistically significant. Exposure to test material did not increase proliferation in the auricular LNs. However, the cell number in the control group was higher than normally observed. Therefore, the calculated SIs are below 1. Dermal application of test material (10%) resulted in a SI of the auricular LNs of 29.3.

 

Effects of exposure to aerosols of test material: Exposure to aerosols of test material (300 ppm) resulted in toxic effects in the mice that were exposed for 360 min/day. After two days of exposure one mouse died and the other mice displayed several signs of distress. These mice were not exposed to test material on the third day. Effects of the two days exposure to test material were assessed on day 5. On the third day two mice died that were exposed for 180 minutes/day for 3 days. The other mice in this group appeared normal. Exposure to test material aerosols resulted in a significant increase of cell number and cell proliferation in the mandibular LNs. This increase was time-dependent, with the exception of the group that was exposed for 360 minutes/day. This group, however, was exposed for two days only. Effects of test material on cell number and cell proliferation were statistically significant for all exposure groups. Test material exposure for 90 minutes/day or longer increased proliferation in the auricular LNs. The mean SI in the auricular LNs was a factor 3-4 higher than in the mandibular LNs, but the variance was very high. Dermal exposure to 10% test material resulted in a SI of 18.8.

 

Test material elicited clearly a higher immune response as indicated by the cellular proliferation in the primary draining lymph node of the nasopharynx. The authors considered that the study provided evidence of respiratory sensitisation.

The reliability of the study cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cutoff value). In addition, no evaluation of irritation to the respiratory tract was made, although Isoeugenol is classified as skin and eye irritant and irritation has been observed in sub-acute inhalation toxicity studies. In the skin LLNA it is required to use dose levels that do not induce excessive levels of irritation. In this study it cannot be determined if the level of irritation was excessive or not, but in some case animals died and showed signs of respiratory distress. Furthermore, there is a potential weakness in the model in that the mandibular lymph node drain the mouth and nasophanrynx but not the tracheal and lung tissues, therefore the relevance to respiratory sensitisation is not proven.

Therefore this study was not used for classification.