Alkanol reaction products with bis(aminoalkyl(C=1~6))carbomonocycle, alkoxy(C=3~8)alkanol(C=2~7) and 2,4-TDI

Endpoint summary

Description of key information

The potential of the substance to induce delayed contact hypersensitivity was investigated using the Local lymph Node Assay (OECD 429, GLP) . The Local lymph Node Assay gave negative results indicating that the substance was not a skin sensitizer.

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:

23 April 2013 - 06 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: the animals of the preliminary test and the animals of the main test were approximately 8 weeks old
- Mean body weight at study initiation: the animals of the preliminary test had a mean body weight of 19.7 g (range: 18.4 g to 20.4 g) and the animals of the main test had a mean body weight of 20.3 g (range: 18.8 g to 21.9 g).
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 22 May 2013 to 04 June 2013

Vehicle:

dimethylformamide

Remarks:

Batch N° SZBC3170V

Concentration:

For the preliminary test the concentrations were 2.5, 5, 10 and 25%, of the test item.
For the main test the concentrations were 0,1, 2.5, 5, 10 and 25% of the test item.

No. of animals per dose:

For the preliminary test: 2 females/dose (no controls)
For the main test: 4 females/dose, 4 females for the negative control and 4 females for the positive control

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil), Dimethylformamide (DMF) was chosen among the other proposed vehicles. A homogenous dosage form preparation was obtained at the concentration of 25% in DMF.
- Irritation: Measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 hours after the last application. In the preliminary test, dryness of the skin was noted on day 6 in both females treated at 5% and in one female treated at 25%. Residual test item was observed in all females treated at 10 and 25% on days 2, 3 and 6. Increase in ear thickness was recorded in one female given the test item at 10% and 25% (+44% and +28%, respectively). This increase was not dose-related and was considered to be due to the residual test item on the ears. The highest concentration retained for the main test was therefore 25%.
- Lymph node proliferation response:not assessed


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The results were expressed as disintegration per minute (dpm) per group. Stimulation indices (SI) were calculated according to the following formula: SI = dpm of treated group / dpm of control group. The test item was considered as a skin sensitizer when the SI for a dose group is higher than or equal to 3. Other relevant criteria such as cellularity (amount of cells in treated group compared to the amount in control vehicle group), radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was prepared in the vehicule at the chosen concentrations. All dosage form preparations were made freshly on the morning of the administration and any unused material was discarded that same day. On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

Positive control substance(s):

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

Statistics:

None

Positive control results:

SI = 9.14 was obtained for the positive control (i.e. HCA 25% in AOO). The experiment was therefore considered valid.
The irritation level was not assessed in the mice treated with the positive control.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

A dose-related increase of the stimulation index was observed at the concentrations of 10 and 25% but it did not exceed the threshold of 3.0 for positive lymphoproliferative response (See table 7.4.1/1). Therefore no notable lymphoproliferation was recorded with the test item at any tested concentration.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per group: Group 3: Vehicle: 2034 Group 4: 1 %: 1227 Group 5: 2.5 %: 1075 Group 6: 5 %: 884 Group 7: 10 %: 2650 Group 8: 25 %: 3499

Table 7.4.1/1: Results of the lymphoproliferative assay

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	1	I	0.60
Test item	2.5	I	0.53
Test item	5	I	0.43
Test item	10	II	1.30
Test item	25	III	1.72
HCA	25	-	9.14

-:        not recorded

I:         non-irritant (increase in ear thickness < 10%)

II:        slightly irritant (increase in ear thickness = 10 to 25%)

III:       irritant (increase in ear thickness = 25%)

HCA:  a-hexyl cinnamaldehyde

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, 98408018 does not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay ( maximum stimulation index = 1.72)

Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce contact hypersensitivity in CBA female mice using the murine Local Lymph Node Assay (LLNA).

This study was conducted in compliance with OECD Guideline No. 429 and the principles of Good Laboratory Practices.

  To assess the irritant potential of the test substance, a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test substance diluted in dimethylformamide by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 2.5, 5, 10 or 25% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Dryness of the skin was noted on day 6 in both females treated at 5% and in one female treated at 25%. Residual test item was observed in all females treated at 10 and 25% on days 2, 3 and 6. Increase in ear thickness was recorded in one females given the test item at 10% and 25% (+44% and +28%, respectively). This increase was not dose-related and was considered to be due to the residual test item on the ears. The highest concentration retained for the main test was therefore 25%.

 In the main test, five groups of four female mice received the test item by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 1, 2.5, 5, 10 or 25% under a dose-volume of 25 µL. One negative control group of four females received the vehicle (dimethylformamide) under the same experimental conditions. Additionally, one positive control group of four females received the positive control, a-hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions.

From day 1 to day 3 then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (³H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph nodes draining the application site was measured by incorporation of³H-TdR. The results are expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

No unscheduled deaths and no clinical signs indicative of systemic toxicity were observed during the study. Body weight of animals was unaffected by the test substance treatment. No local reactions were observed in any animals. Residual test item was observed in one out of four females treated at 5% on day 6 and in all females treated at 10% and 25% on days 2, 3 and 6. A notable increase in ear thickness (+33%) was observed in females treated at 25%. This was correlated with the presence of residual test item.

The SI of the positive control was 9.14; this experiment was therefore considered valid. A dose-related increase of the stimulation index was observed at the concentrations of 10 and 25 % (1.30 and 1.72 respectively) but it did not exceed the threshold of 3.0 for positive lymphoproliferative response. Therefore no notable lymphoproliferation was recorded with the test item at any tested concentration.

It was concluded that the test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A study was performed according to OECD guideline No. 429 and in compliance with good Laboratory Practices. The test substance was administered to mice and the Lymph node proliferative responses were measured as described by Kimber and Dearman (1991). No clinical signs and no mortality were observed during the study. Furthermore, no irritation of the skin was noted following the application of the test item up to the highest tested concentration (25%). The LLNA gave negative results, as the Stimulation Index (SI) was not higher than 1.72 whatever the tested concentrations. Under the test conditions, the test substance was not considered as a dermal sensitizer in the murine Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The maximum Stimulation Index (SI) found in the study was 1.72. According to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, the substance is not classified as the SI is lower than 3%.