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Description of key information

Skin sensitisation: Ulrich et al., 2001. Comparable to the OECD TG 406: Not sensitising

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
other: according to Ulrich, P. et al. 1998: Toxicology 125, 149-168
Version / remarks:
modified protocol of the LLNA
Principles of method if other than guideline:
- Principle of test: DMF was used as vehicle in a series of experiments with a panel of standard contact (photo)allergens and (photo)irritants in the course of the validation of a two-tiered murine LLNA.
- Short description of test conditions: Groups of 6 mice were used, test chemical solutions were applied on three consecutive days to the dorsum of both ears. If testing for photo reactivity was required, mice were exposed to UVA light at a dose of 10 J/cm (5 mW/cm2 for33 min) immediately after test chemical application. Auricular LNs draining the ear tissue were excised 24 h after the initial exposure.

For the comparison of the induction and challenge responses, mice were treated on the shaved back with 50 µL of test chemical or vehicle alone on three consecutive days (induction phase treatment). Mice were challenged 12 days after the initial induction phase exposure with 25 µL of test chemical or vehicle on the dorsum of both ears for a further 3 days (challenge phase treatment).Auricular LNs draining the ear tissue were excised 24 h after the initial exposure.

Tier I: A wide range of concentrations of test chemical solutions were applied. Ear weights were determined to correlate chemical induced skin irritation with the ear-draining lymph node activation potential. The induction and challenge responses were compared.
Tier II: LLNA protocol was used to finally differentiate between true irritants and contact allergens.

- Parameters analysed / observed: Determination of ear weights, LN weights and LN cell counts, skin irritation (analytical scales as described previously (Ulrich et al. 1998)).
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-8 weeks old
Vehicle:
unchanged (no vehicle)
Concentration:
Tier I: A wide range of concentrations of test chemical solutions were applied
No. of animals per dose:
Six female per group
Details on study design:
Groups of mice received 25 µL of test chemical solution or vehicle to the dorsum of both ears on three consecutive days. If testing for photo reactivity was required, mice were exposed to UVA light at a dose of 10 J/cm (5 mW/cm² for 33 min) immediately after test chemical application using a Psorisan 900 H1 lamp (Dr. Honle, Medizintechnik, Munich, Germany) with a UVA (320-400 nm) emission maximum at 380 nm. The use of the H1 ilter(Dr. Honle) reduced the amount of UVB (<320 nm) to an undetectable level, which was checked with a UVA/UVB meter (Dr. Honle). Auricular LNs draining the ear tissue were excised 24 h after the initial exposure.

For the comparison of the induction and challenge responses, mice were treated on the shaved back with 50 µL of test chemical or vehicle alone on three consecutive days (induction phase treatment). Mice were challenged 12 days after the initial induction phase exposure with 25 µL of test chemical or vehicle on the dorsum of both ears for a further 3 days (challenge phase treatment). LNs were excised 24 h after the initial challenge phase treatment.

Determination of ear weights, LN weights and LN cell counts

Using analytical scales as described previously (Ulrich et al. 1998). In addition, a circular piece (0.28 or 0.5 cm2) was punched from the apical ear area and weighed on analytical scales. For the determination of individual LN cell counts, single-cell suspensions from LN pairs from individual animals were prepared by mechanical tissue disaggregation through a sterile stainless steel gauze in 1 ml PBS (Ca2+/Mg2 + -free) containing 0.5 % BSA. Cell counts were determined by conductometry (CASY cell counter; Scharfe System, Reutlingen, Germany) gating on a particle diameter above 4.88 µm.
Positive control substance(s):
other: dinitrochlorobenzene (DNCB)
Statistics:
A one-way analysis of variance was performed for the group effect. A quantile plot was used to visually judge the normality of the residuals. In some cases, a transformation was applied and/or outliers were omitted in order to improve the normality of the residuals. A multiple comparison method was further applied using the methods of Tukey (Hayter 1989), Sidak (Sidak 1967) and Dunnett (Dunnett 1964). The Dunnett test compares every treated group with the control group while the other two methods can be used to compare each group with every other group. The multiple comparison provides an estimation of the difference in the expected response between the two compared groups, the standard error of the response and a lower and an upper confidence limit for the difference. If the two limits do not include zero the difference is significantly different from zero at the 5 % level. By repeating the method for 1 % and 0.1 %, the strength of the significance is determined. S-Plus software (S-Plus 5 for Unix, 1998, MathSoft, Seattle, Wash.) was used forthe computations. The three methods (Tukey, Sidak and Dunnett) were used adaptively, i.e. in every case the most sensitive of the methods was used.
Parameter:
SI
Test group / Remarks:
6 female mice
Remarks on result:
not determinable because of methodological limitations
Remarks:
First experiment: slight ear-draining lymph node activation as expressed by increased weights and cell counts (experiment with DNCB). However, this observation was not reproducible in a second experiment (when DMF was tested as vehicle for eugenol and as vehicle alone in comparison to the respective untreated control group).
Parameter:
SI
Test group / Remarks:
6 female mice
Remarks on result:
not determinable because of methodological limitations
Interpretation of results:
other:
Remarks:
EU GHS criteria not met
Conclusions:
Topical treatment of mice with the vehicle N,N-dimethylformamide led to slight ear-draining lymph node activation as expressed by increased weights and cell counts in comparison to the untreated animals. However, this observation was not reproducible in a second experiment (when DMF was tested as vehicle for eugenol and as vehicle alone in comparison to the respective untreated control group).
Executive summary:

Study design

This non-GLP in vivo study was conducted following a modified LLNA-protocol with respectable restrictions (Ulrich et al., 1998, Toxicology 125, 149 -168).

DMF was used as vehicle in a series of experiments with a panel of standard contact (photo)allergens and (photo)irritants in the course of the validation of a two-tiered murine LLNA (Ulrich et al., 2001). Groups of 6 female BALB/C strain mice (6-8 weeks old) were used. During tier I a wide range of concentrations of test chemical solutions or vehicle (volume: 25 µL) were applied on three consecutive days to the dorsum of both ears. Mice were killed 24 hours after the last application to determine ear and local lymph node weights and lymph node cell counts. Ear weights were determined to correlate chemical induced skin irritation with the ear-draining lymph node activation potential. For comparison of the induction and challenge responses, mice were treated on the shaved back with 50 µL of test chemical or vehicle alone on three consecutive days (induction phase treatment). Then mice were challenged 12 days after the final induction phase exposure with 25 µL of test chemical or vehicle on the dorsum of both ears for a further 3 days (challenge phase treatment). Lymph nodes were excised 24 hours after the final challenge phase treatment. A tier II LLNA protocol was used to finally differentiate between true irritants and contact allergens. To investigate the impact of different vehicles on the primary response induced by two contact allergens, i.e dinitrochlorobenzene (DNCB) at 0.5 % and by eugenol at 35 %, DAE433, DMSO, DMF and acetone /oil olive (AOO) were used. Both contact allergens were compared either to the untreated control (aqua bidest) or to the corresponding vehicle control.

Results and conclusion

Topical treatment of mice with the vehicle DMF led to slight ear-draining lymph node activation as expressed by increased weights and cell counts in comparison to the untreated animals. However, this observation was not reproducible in a second experiment (i.e. when DMF was tested as vehicle for eugenol and as vehicle alone in comparison to the respective untreated control group).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

This non-GLP in vivo study was conducted following a modified LLNA-protocol with respectable restrictions (Ulrich et al., 1998, Toxicology 125, 149 -168).

DMF was used as vehicle in a series of experiments with a panel of standard contact (photo)allergens and (photo)irritants in the course of the validation of a two-tiered murine LLNA (Ulrich et al., 2001). Groups of 6 female BALB/C strain mice (6-8 weeks old) were used. During tier I a wide range of concentrations of test chemical solutions or vehicle (volume: 25 µL) were applied on three consecutive days to the dorsum of both ears. Mice were killed 24 hours after the last application to determine ear and local lymph node weights and lymph node cell counts. Ear weights were determined to correlate chemical induced skin irritation with the ear-draining lymph node activation potential. For comparison of the induction and challenge responses, mice were treated on the shaved back with 50 µL of test chemical or vehicle alone on three consecutive days (induction phase treatment). Then mice were challenged 12 days after the final induction phase exposure with 25 µL of test chemical or vehicle on the dorsum of both ears for a further 3 days (challenge phase treatment). Lymph nodes were excised 24 hours after the final challenge phase treatment. A tier II LLNA protocol was used to finally differentiate between true irritants and contact allergens. To investigate the impact of different vehicles on the primary response induced by two contact allergens, i.e dinitrochlorobenzene (DNCB) at 0.5 % and by eugenol at 35 %, DAE433, DMSO, DMF and acetone /oil olive (AOO) were used. Both contact allergens were compared either to the untreated control (aqua bidest) or to the corresponding vehicle control.

Topical treatment of mice with the vehicle DMF led to slight ear-draining lymph node activation as expressed by increased weights and cell counts in comparison to the untreated animals. However, this observation was not reproducible in a second experiment (i.e. when DMF was tested as vehicle for eugenol and as vehicle alone in comparison to the respective untreated control group).

DMF was also negative in Guinea Pig Maximization Test (Bainova, 1985, cited in BUA-Stoffdossier N,N-Dimethylformamid, Stand 04/91).

Justification for selection of skin sensitisation endpoint:
The best study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In the sub-chronic inhalation study (Lynch et al., 2003), the animals were exposed to DMF by whole body inhalation exposure at 0, 50, 100, 200, 400, or 800 ppm, 6h/day, 5days/week, for 13 weeks. DMF was mildly irritating to rats exposed at 400 and 800 ppm, evidenced by occasional nasal and ocular discharges. Organs and tissues from high dose group animals and from the controls were examined histopathologically and examined for gross lesions. Under these organs were also lungs, main stem bronchi and tracheas. Microscopically, no lesions, associated with exposure to DMF, were found in these organs. DMF was not sensitizing to the respiratory tract in the test animals.


Short description of key information:
Lynch et al., 2003. Thirteen-Week Inhalation Toxicity of N,N-Dimethylformamide in F344/N Rats and B6C3F1 Mice. Publication. Comparable to the OECD guideline 413.

Justification for selection of respiratory sensitisation endpoint:
No study is selected since the information has been taken from a repeated inhalation study summarized under section 7.5.2.

Justification for classification or non-classification

The classification is not warranted according to the criteria of Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008.