Dimethyl propylphosphonate

Endpoint summary

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Guideline adopted 22 July 2010; Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

Deviations:

yes

Remarks:

IDMS LLNA: Measurement of cell counts instead of radioactive labelling. In addition, ear swelling and ear weights are determined to discriminate the irritating potential from the sensitizing potential of the test substance.

Principles of method if other than guideline:

This study is performed according to OECD TG 429. As stated in OECD TG 429 besides the classical radioactive method ‘other endpoints for assessment of the number of proliferating cells may be employed’ as so-called ‘me-too’ tests, if the required performance standards are fulfilled, they are ‘based on similar scientific principles and measure or predict the same biological or toxic effect’ and they are validated.
Here, an alternative method is used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes (IMDS LLNA; Integrated Model for the Differentiation of Skin Reactions). In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties. Information on validation of the IMDS LLNA and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).
In the IMDS LLNA stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify skin sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).
- Vohr, H.-W., Blümel, J., Blotz, A., Homey, B. and Ahr, H.J. An intra-laboratory validation of IMDS: Discrimination between (Photo) Allergic and (Photo) Irritant Skin Reactions in Mice. Arch. Toxicol., 73, 501-509 (2000).

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 26-31 g
- Housing: singly during study
- Diet ad libitum
- Water ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

Group 1 Vehicle (A/OO = acetone/olive oil)
Group 2 10% dimethyl propylphosphonate (in A/OO)
Group 3 30 % dimethyl propylphosphonate (in A/OO)
Group 4 100 % dimethyl propylphosphonate
Group 5 40 % Alpha Hexyl Cinnamic Aldehyde (in A/OO)

No. of animals per dose:

6 animals/test item group and 6 control animals

Details on study design:

The test item in the formulation, the positive control in the formulation or the vehicle were applied epicutaneousely onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days. The volume administered was 25 µl/ear. The used concentrations were based on the experience with this test system and the properties of the test item.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application. The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline
Investigations:
- weight of lymph nodes
- cell counts in lymph nodes
- stimulation index is calculated by dividing the absolute number of weight or cell count of the substance-treated lymph nodes by the vehicle treated ones
- ear swelling
- ear weight
- body weight

Positive control substance(s):

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

Statistics:

When it was statistically reasonable, the values from treated groups were compared with those from the control group by one-way Analysis of Vartiance (ANOVA) when the variances are considered homogenous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogeneous a non-parametric Kruskal-Wallis test has been used at significance levels of 5%. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Sheffe's method, which can be used for both equal and unequal sample sizes.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

A modified Local Lymph Node Assay (IMDS) was carried out in mice. The modifications refer to the measurement of cell proliferation by cell counting instead of radioactive labeling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes, as also recommend in the update of OECD TG 429. Direct LLNA (NMRI mice, female, 6 animals/group) Groups Weight index Cell count index (index of mean +/-SD in %) Gr.1 1.00 +/- 26.52 1.00 +/- 26.46 Gr.2 1.06 +/- 31.38 1.06 +/- 38.46 Gr.3 0.91 +/- 16.23 0.91 +/- 23.67 Gr.4 0.97 +/- 43.54 0.98 +/- 65.67 Gr.5 1.80 +/- 29.30 2.16 +/- 36.11 Ear swelling (NMRI mice, female, 6 animals/group, in 0.01 mm) Groups day 1 day 4 Index day 4 (mean +/- SD in %) Gr.1 17.50 +/- 3.85 19.08 +/- 5.22 1.00 Gr.2 17.25 +/- 2.62 18.83 +/- 5.92 0.99 Gr.3 17.50 +/- 2.98 18.83 +/- 5.47 0.99 Gr.4 17.33 +/- 2.84 18.58 +/- 5.36 0.97 Gr.5 17.42 +/- 4.55 22.00 +/- 7.99 1.15 Ear weight (NMRI mice, female, 6 animals/group, in mg per 8 mm diameter punch) Groups day 4 Index day 4 (mean +/- SD in %) Gr.1 11.69 +/- 6.23 1.00 Gr.2 12.36 +/- 9.60 1.06 Gr.3 12.13 +/- 6.90 1.04 Gr.4 11.36 +/- 8.03 0.97 Gr.5 14.17 +/- 7.12 1.21 Group 1 Vehicle (A/OO = acetone/olive oil) Group 2 10% dimethyl propylphosphonate (in A/OO) Group 3 30 % dimethyl propylphosphonate (in A/OO) Group 4 100 % dimethyl propylphosphonate Group 5 40 % Alpha Hexyl Cinnamic Aldehyde (in A/OO)

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Modified LLNA. Measurement of cell counts instead of radioactive labelling.

Stimulation indices (weight and cell counts; ear swelling and ear weight)

It has to be clarified that the "positive levels" mentioned above are exclusively defined for the NMRI outbreed mice used for this study. Such positive limits have to be calculated for each strain of mice individually.

Based on results obtained in validation studies and general experiences with this test system groups of mice were treated with vehicle, 10 %, 30% or 100% dimethyl propylphosphonate in acetone/olive oil (A/OO).

The NMRI mice did not show an increase in the stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test item dimethyl propylphosphonate.

The "positive level", which is 1.4 for the cell count index, was never reached or exceeded in any dose group.

The "positive level" of ear swelling, which is 2xl0E-2 mm increase, i.e. about 10% of the control values, has not been reached or exceeded in any dose group.

No substance specific effects were determined for ear weights either.

After treatment with Alpha Hexyl Cinnamic Aldehyde (group 5) the NMRI mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to control animals, which are of statistical significance. The "positive level", which is 1.4 for cell count indices, has clearly been exceeded.

The "positive level" of ear swelling, which is 2x 10E-2 mm increase, i.e. about 10 % of the control values, has been exceeded in the positive control group. This increase is of statistical significance. A significant increase compared to vehicle treated animals regarding ear weights was detected, too.

 

Body weights

The body weights of the animals were not affected by any treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

The LLNA was negative. Dimethyl propylphosphonate shows no sensitizing potential in the modified Local Lymph Node Assay (IMDS) in female NMRI mice after dermal application of up to and including a 100 % concentration of dimethyl propylphosphonate.

Executive summary:

The modified Local Lymph Node Assay (IMDS) was performed in 2012 on 24 female NMRI mice of the strain HsdWin:NMRI (6 animals/test item group and 6 control animals) to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of the test item dimethyl propylphosphonate.

A concurrent control of 6 animals treated with Alpha Hexyl Cinnamic Aldehyde (alpha-HCA) was included.

The study was conducted according to OECD Guidelines No. 429 and No. 406, EC Guideline 2004/73/EC (29th Adaptation of Guideline 67 /548/EEC, B.42)/Health Effects Test Guideline and OPPTS 870.2600 (EPA) with the following test item concentrations:

Test item: 0% (vehicle control), 10%, 30% and 100%.

Positive control: 40% Alpha Hexyl Cinnamic Aldehyde

The test item and the positive control were formulated in acetone/olive oil ( 4:1) (A/OO) to yield a solution.

Compared to vehicle-treated animals, none of the parameters measured in the substancetreated groups, i.e. cell counts and weights of the draining lymph nodes, ear weights and ear swelling, reached or exceeded the "positive levels" defined for this assay. These

results show that there is no indication for a skin sensitizing effect after administration of a concentration up to and including 100 % dimethyl propylphosphonate in this test system.

In conclusion, these results show that the test item dimethyl propylphosphonate has no sensitizing potential in mice after dermal application of up to and including a 100 % concentration. No indication for a non-specific (irritant) activation was detected, too. Therefore, the concentration of 100 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization. These results are verified by the comparison with the results of the positive control group (Alpha Hexyl Cinnamic Aldehyde).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Compared to vehicle-treated animals, none of the parameters measured in the substance treated groups, i.e. cell counts and weights of the draining lymph nodes, ear weights and ear swelling, reached or exceeded the "positive levels" defined for this assay. These results show that there is no indication for a skin sensitizing effect after administration of a concentration up to and including 100% dimethyl propylphosphonate in this test system.

In conclusion, these results show that the test item dimethyl propylphosphonate has no sensitizing potential in mice after dermal application of up to and including a 100% concentration. No indication for a non-specific (irritant) activation was detected, too. Therefore, the concentration of 100% turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.

Migrated from Short description of key information:
A modified Local Lymph Node Assay (IMDS) was performed on 24 female NMRI mice (6 animals/test item group and 6 control animals) to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of the test item dimethyl propylphosphonate.
A concurrent control of 6 animals treated with Alpha Hexyl Cinnamic Aldehyde was included.
The study was conducted according to OECD Guidelines No. 429 and No. 406, EC Guideline 2004/73/EC (29th Adaptation of Guideline 67 /548/EEC, B.42)/Health Effects Test Guideline and OPPTS 870.2600 (EPA) with the following concentrations: Dimethyl propylphosphonate: 0% (vehicle control), 10%, 30% and 100%. Positive control: 40% Alpha Hexyl Cinnamic Aldehyde

Justification for selection of skin sensitisation endpoint:
the only available study is used

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No data

Migrated from Short description of key information:
No data

Justification for classification or non-classification

Due to the negative result in the LLNA a classification is not justified.