Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and Ethanol, 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-

Endpoint summary

Description of key information

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, the substance was considered to be a skin sensitiser, as the SI appeared to be ≥ 3 when tested up to 100%.

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:

February 27th - March 18th, 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

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

issued 19 May 2011

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Young adult females (approx. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (20-23 g).
- Housing: Animals were group housed in labeled Makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 March 2013 - 18 March 2013

Vehicle:

dimethylformamide

Remarks:

or unchanged (100%)

Concentration:

25, 50, 100%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
Three test substance concentrations were tested: 25, 50 and 100 % concentration (% w/w). Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels. Correction of the purity/composition of the test substance is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be applied.

Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing). Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy was conducted on the animals that were found dead or sacrificed for humane reasons.

Positive control substance(s):

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

Statistics:

Not performed.

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

Key result

Parameter:

SI

Value:

3.2

Test group / Remarks:

Test group 25 %

Key result

Parameter:

SI

Value:

4.7

Test group / Remarks:

Test group 50 %

Key result

Parameter:

SI

Value:

10.8

Test group / Remarks:

Test group 100 %

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 2008, 2925 and 6725 DPM respectively. The mean DPM/animal value for the vehicle control group was 624 DPM.

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined at 25 and 50%. Both animals at 100% showed very slight erythema on Day 5 only.

Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Greasy test substance remnants were present on the dorsal surface of the ears of both animals at 100% (Days 2 and 3), which did not hamper scoring of the skin reactions. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

Other results - main study:

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

No irritation of the ears was observed in any of the animals examined.

Greasy test substance remnants were present on the dorsal surface of the ears of all animals at 50 and 100% (Days 2 and/or 3), which did not hamper scoring of the skin reactions.

The lymph nodes were larger in size in animals at 50 and 100%. The largest auricular lymph nodes were found in the higher dose group. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Category 1 classification according to EC Regulation 1272/2008.

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, the substance was considered to be a skin sensitiser, as the SI appeared to be ≥ 3 when tested up to 100%. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 25%.

Executive summary:

An LLNA skin sensitisation assay was performed according to OECD/EC guidelines and GLP principles. The test substance was applied at concentrations of 25, 50 or 100%. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. No irritation of the ears was observed in any of the animals examined. The lymph nodes were larger in size in animals at 50 and 100%. The largest auricular lymph nodes were found in the higher dose group. No macroscopic abnormalities of the surrounding area were noted in any of the animals. The SI values calculated for the substance concentrations 25, 50 and 100% were 3.2, 4.7 and 10.8 respectively.

These results show that the test substance elicits an SI≥3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 25%. Based on these data, the substance is classified for skin sensitisation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

An LLNA skin sensitisation assay was performed according to OECD/EC guidelines and GLP principles. The test substance was applied at concentrations of 25, 50 or 100%. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. No irritation of the ears was observed in any of the animals examined. The lymph nodes were larger in size in animals at 50 and 100%. The largest auricular lymph nodes were found in the higher dose group. No macroscopic abnormalities of the surrounding area were noted in any of the animals. The SI values calculated for the substance concentrations 25, 50 and 100% were 3.2, 4.7 and 10.8 respectively.

These results show that the test substance elicits an SI≥3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 25%.

Justification for selection of skin sensitisation endpoint:
a reliable study is available, study was performed according to OECD guideline and GLP principles (Klimisch score = 1).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is classified for skin sensitisation according to Regulation (EC) No 1272/2008.