4-morpholinecarbaldehyde

Endpoint summary

Description of key information

Murine Local Lymph Node Assay: positive (BASF, 2011)

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

Study period:

2011-06-15 to 2011-06-29 (exp. period)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study OECD 429 and GLP compliance

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS

- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, Netherlands
- Age at study initiation: 9-10 weeks old
- Weight at study initiation: 17.5 - 21.1 g
- Housing: Single (pre-test); Group (main study)
- Diet: Pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 45 - 65
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

1) 25 %
2) 50 %
3) 100 %

No. of animals per dose:

5 mice

Details on study design:

RANGE FINDING TEST
For concentration selection one mouse was treated at 75% in acetone olive oil (4:1 v/v) and a second mouse was treated at 100 % (undiluted) for 3 consecutive days. Signs of systemic toxicity, body weights, ear weights, ear thicknuess abd ear erythema were assessed.
- Compound solubility: soluble in acetone olive oil (4:1 v/v)
- Irritation: both animals showed very slight to well defined eryhema with an increase in ear swelling. Undiluted test item (100 %) was selected as highest concentration for the main study as it did neither induce signs of systemic toxicity nor exessive skin irritation.

MAIN STUDY
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 % (v/v), and 100 % of the 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 of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.

Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 20.5 µCi of 3HTdR (equivalent to approximately 82.0 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium. The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared and after several washing steps the level of 3HTdR incorporation was measured using a beta-scitillation counter.
Furthermore, the lymph node cell count was determined.

After the lymph nodes were excised, both ears of all mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
In addition 3HTdR incorporation, lymph node weights, lymph node cell count, ear weights and ear thickness, the mortality and clinical signs of toxicity were assessed in this study.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for body weight and for the radioactive disintegrations per minute (DPM) values (group mean DPM ± standard deviation) as well as for the ear weights, lymph node weights and lymph node cell counts.

Statistical analysis was conducted to assess whether the difference was statistically significant between test 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.
For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance (One-Way-ANAOVA) was used as statistical method.
In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test or Dunn’s test. Statistical significance was set at the 5 %t level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers.
Also, the ANOVA (Dunnett-test) was conducted on 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 (vehicle) group.
Both biological and statistical significance were considered together.

Positive control results:

In the periodic positive control experiment with alpha-Hexylcinnamaldehyde performed in May 2011 the EC3 (= estimated concentration for a S.I. of 3) was calculated to be 10.4 %.

Parameter:

SI

Value:

1

Test group / Remarks:

0%

Remarks on result:

other: negative control

Parameter:

SI

Value:

2.07

Test group / Remarks:

25%

Parameter:

SI

Value:

4.36

Test group / Remarks:

50%

Parameter:

SI

Value:

2.91

Test group / Remarks:

100%

Viability / Mortality:

No deaths occurred during the study period.

 

Clinical Signs:

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study

 

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 increase in lymph node weight was observed at all tested concentrations (25, 50, and 100%) in comparison to the vehicle control group (p<0.05).

For BALB/c mice, a cut off-value for the lymph node cell count index of 1.55 was reported for a positive response. The index determined for the high dose group (1.53) was close, the index determined for the mid dose group (1.89) exceeded this threshold. A statistically significant increase in lymph node cell count was not observed in comparison to the vehicle control group.

 

Ear Weights:

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weight was observed for the mid and high dose group (50 and 100 %) 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. The ear weight indices determined for 50 and 100% were both slightly above this threshold (with indices of 1.19, each), indicating the presence of local skin irritation. The ear weight index determined for the lowest dose group (1.1) indicates a borderline positive response regarding ear skin irritation. However, the measured percentage of increase in ear weight (10.2 %, 18.8 %, and 18.6 %, at the low, mid and high dose, respectively) did not exceed the threshold value of 25 % for excessive local skin irritation.

Interpretation of results:

sensitising

Conclusions:

In this study Stimulation Indices (S.I.) of 2.07, 4.36, and 2.91 were determined with the test item at concentrations of 25, 50% (v/v) in acetone:olive oil (4+1 v/v), and 100% (undiluted test item), respectively, and an EC3 value of 35.2% (v/v) was calculated. The test item showed an unusual dose-response relationship with a decreased S.I. at the highest tested concentration (100% of the undiluted test item) that might be due to different properties of the undiluted test item and the test item solution in acetone:olive oil (4+1 v/v) , e.g. immunosupression when applied undiluted.. On the basis of the present data, the test item has to be classified as a sensitiser.

Executive summary:

It is concluded that N-formylmorpholine exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen and should be classified as a sensitiser according to Regulation 1272/2008/EC (GHS cat. 1B). (BASF, 2011)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In the key dermal sensitisation study with N-formylmorpholin (Harlan CCR, 2011; 58V0173/97X001) groups of 5 female CBA/CaOlaHsdmice each were treated with 25 % or 50 % w/w preparations of the test item in acetone:olive oil or 100 % (undiluted test item) using the Local Lymph Node Assay. The test item showed a skin sensitizing effect under the test conditions chosen based on statistically significant and biologically relevant increases. The threshold concentration for sensitization induction was between 25 % and 50 %. The EC 3 for ³H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 35.2 %.

Short description of key information:
The test item N-formylmorpholine was found to be a skin sensitiser in the Local Lymph Node Assay.

Justification for selection of skin sensitisation endpoint:
The key study was selected (GLP and Guideline study).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the key sensitisation study, N-formylmorpholine is subjected to classification and labelling as follows:

Skin sensitizer Cat 1 B / H317 (may cause an allergic skin reaction) according to Comission Regulation (EU) No 286/2011 representing the second ATP to Regulation 1272/2008/EC.