Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-17 and C17 unsatd. alkyl) derivs. and sodium hydroxide and 2-propenoic acid

Endpoint summary

Description of key information

Sensitizing (Cat. 1B) EC3 = 30.6% (NESIL = 7650 µg/cm²); OECD TG 429, in vivo, LLNA, mouse (GLP, RL1); read-across: Amphopropionate C8

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:

2007-06-06 to 2007-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2004)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2002)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 22 - 27 g
- Housing: Individual housing in labeled Macrolon cages (Ml type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNlFF® Spezialdiaten GmbH, Soest, Germany).
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7- 23.0°C
- Humidity (%): 43 - 78%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Concentration:

0%, 25%, 50%, 100%

No. of animals per dose:

5

Details on study design:

PRELIMINARY IRRITATION STUDY
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 2) at the highest. A series of two test substance concentrations was tested, selected from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1% and if needed further lower concentrations using the same steps. The highest concentration, selected from this series, was the undiluted test substance. The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the ear was cleaned of residual test substance with tap water/vehicle and the irritation was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Induction (days 1,2 and3): The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.
- Treatment (day 6): Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 pCi of 3H-methyl thymidine.
After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital (0.2 mL/animal). The draining (auricular) lymph node of each ear was excised.
The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

TISSUE PROCESSING AND MEASUREMENTS:
- Tissue processing for radioactivity (day 6): A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation
through stainless steel gauze (diameter 125 pm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4° C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) at 4° C during the night.
- Radioactivity measurements (day 7): Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
- 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 sensitiser, based on the test guideline and recommendations done by ICCVAM.

Positive control substance(s):

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

Positive control results:

The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.The SI values calculated for the substance concentrations 5, 10 and 25% were 1.3, 1.5 and 5.5 respectively. An EC3 value of 15.6% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%.

Parameter:

SI

Value:

1.9

Test group / Remarks:

25%

Parameter:

SI

Value:

6.8

Test group / Remarks:

50%

Parameter:

SI

Value:

8.5

Test group / Remarks:

100%

Parameter:

EC3

Value:

30.6

PRELIMINARY IRRITATION STUDY

No irritation was observed in any of the animals examined. The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in the table. Based on the results, the highest test substance concentration selected for the main study was a 100% concentration.

MAIN STUDY

- Skin reactions/Irritation: No skin reactions were observed in any of the animals examined.

- Macroscopy of the auricular lymph nodes and surrounding area: The majority of nodes were considered normal in size, except for enlarged nodes of two animals at 50% and two animals at 100%. No macroscopic abnormalities of the surrounding area were noted.

- Body weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study

- Toxicity and Mortality: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The SI values calculated for the substance concentrations 25, 50 and 100% were 1.9, 6.8 and 8.5 respectively. These results indicate that the test substance could elicit an SI ≥ 3 and Amphopropionate C8 is therefore regarded as skin sensitiser. The data showed a dose-response and an EC3 value of 30.6% was calculated.

Executive summary:

In a local lymph node assay according to OECD guideline 429 (2002) and EU Method B.42 (2004) Amphopropionate C8 dissolved in dimethylformamide was assessed for its possible contact allergenic potential using test item concentrations of 25, 50 and 100%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.9, 6.8, and 8.5 were determined with the test item at concentrations of 25, 50, and 100% in dimethylformamide, respectively. These results indicate that the test substance could elicit an SI ≥ 3 and Amphopropionate C8 is therefore regarded as skin sensitiser under the conditions of this study. The data showed a dose-response and an EC3 value of 30.6% was calculated.

Based on these results Amphopropionate C8 is classified as Category 1B based on CLP, EU GHS (Regulation (EC) No 1272/2008).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A dermal sensitisation study conducted with the target substance Amphopropionates C12 -18 is available, which was, however, not reliable and is used only as supporting information. A reliable study conducted with the closely related source substance Amphopropionate C8 is available. A justification for read-across is given below.

 

In a local lymph node assay according to OECD guideline 429 (2002) and EU Method B.42 (2004) Amphopropionate C8 dissolved in dimethylformamide was assessed for its possible contact allergenic potential using test item concentrations of 25, 50 and 100%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.9, 6.8, and 8.5 were determined with the test item at concentrations of 25, 50, and 100% in dimethylformamide, respectively. These results indicate that the test substance could elicit an SI ≥ 3 and Amphopropionate C8 is therefore regarded as skin sensitiser under the conditions of this study. The data showed a dose-response, and an EC3 value of 30.6% was calculated.

From that a NESIL (No Expected Sensitisation Induction Level) of 7650 µg/cm² was derived.

 

Supporting data are available for the target substance itself:

In a dermal sensitization study according to OECD guideline 406 (1981) with Amphopropionates C12 -18 (solid content: 39 – 41%) 20 Pirbright-Hartley guinea pigs were tested using the Guinea pig maximization test according of Magnusson and Kligman.

Based on the results of the range finding study for intradermal and epicutaneous induction procedure test substance concentrations of 10 % and 1 % in water were used, respectively. The test substance concentration for the challenge procedure was 1 % in water.

The challenge after 24 hours (first reading) indicated that 18/20 (90%) test animals treated with the test substance Amphopropionates C12 -18 and 11/20 (55%) negative control animals showed a positive skin (slight erythema) reaction. 48 hours after challenge application (second reading) 45% of test animals and 30% of negative control animals showed a slight erythema reaction. 10/20 test animals and 11/20 negative control animals developed a slight desquamation after 48 hours, respectively.

The tendency of most of the responses at the first reading implies to be more likely irritant than skin sensitive. A verification of a sensitizing reaction by a rechallenge as recommended by OECD TG 406 was not performed. In addition the chosen concentration for dermal induction was the highest non-irritating concentration and not a concentration which causes mild-to-moderate skin irritation, as required by the OECD TG 406.

However, based on the ambiguous results a final interpretation is not possible.

Furthermore, no positive control substance was tested in parallel and no periodically performed sensitivity and reliability check of the experimental technique is reported. Thus, this study is rated as Klimisch 3 (not reliable). 

 

There is no data gap for skin sensitisation. No human data are available. However, there is no reason to believe that these results from rabbit would not be applicable to humans.

 

Justification for read-across

For details on substance identity and detailed toxicological profiles, please refer also to the general justification for read-across given at the beginning of the CSR and attached as pdf document to IUCLID section 13.

This read-across approach is justified based on structural similarities as well as on a similar toxicological profile. The target and source substances contain the same functional groups. Thus a common mode of action can be assumed.

 

Structural similarity and functional groups

The target substance Amphopropionates C12-18 is manufactured from fatty acids (C12-18, C18unsatd.) and aminoethylethanolamie (AEEA) to form 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-C17 odd-numbered, C17unsatd. alkyl) derivs. This is further reacted with 2-propenoic acid in the presence of sodium hydroxide (alternatively, sodium 2-propenoate can be used) and water. The molar relation between 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-C17, C17unsatd. alkyl) derivs. and 2-propenoic acid is somewhat below 1:1. Most of the excess 2-propenoic acid is stripped off by distillation. However, a small amount remains in the aqueous solution.

 

The source substance Amphopropionate C8 is manufactured from capric acid and aminoethylethanolamine (AEEA) to form 1-(2-Hydroxyethyl)-2-Heptylimidazoline. Excess AEEA is removed from the reaction mixture by distillation at elevated temperature. In a further step 2-propenoic acid is added to form Amphopropionate C8. Most of the excess 2-propenoic acid is stripped off by distillation. However, a small amount remains in the aqueous solution.

 

Differences

Chain length:

The source substance Amphopropionate C8 contains shorter C chains, whereas the major C chain in the target substance is C12.

In general the absorption declines with increasing alkyl chain length (Ramirez et al. 2001). Therefore the source substance with the shorter alkyl chain length is assumed to represent a worst-case scenario due to higher absorption rates than the target substance.

 

Degree of unsaturation:

In contrast to the source substance Amphopropionate C8, the target substance Amphopropionates C12-18 contains some amounts of unsaturated C18 chains.

An increase in the degree of unsaturation may lead to a slightly higher irritation potential (HERA, 2002; Stillman, 1975; Aungst, 1989). Apart from that, fatty acids irrespective of their degree of unsaturation are in general non-toxic. Irritation studies are available for the target substance itself, thus, for other endpoints, this difference in composition is of no toxicological relevance.

 

The provided structural similarities and impurity profiles support the proposed read-across hypothesis with high confidence.

 

Comparison of sensitisation data

 

	Target substance	Source substance
	Amphopropionates C12-18	Amphopropionate C8
Skin sensitisation	sup_Skin sensitisation: 93820-52-1_8.3_REWO_1988_OECD 406   OECD TG 406, GPMT   Positive reactions in 18/20 (90 % ) test animals and 11/20 (55 %) negative control animals   Ambiguous (but no rechallenge)   3 (not reliable, methodological deficiencies), GLP	key_RA_Skin sensitisation_64265-45-8_8.3_Evonik_2007_OECD429   mouse, OECD TG 429, LLNA   Sensitising, Cat. 1 B (EC3 =30.6%)   1 (reliable without restriction), GLP

 

A study on the skin sensitisation potential of Amphopropionates C12 -18 is available. However, based on the ambiguous results a final interpretation was not possible. Based on the results from a study conducted with the closely related source substance Amphopropionate C8, the target substance Amphopropionates C12 -18 is considered to be a dermal sensitiser (EC3=30.6%; classified as Category 1B).

 

Quality of the experimental data of the analogues:

The available data are adequate and sufficiently reliable to justify the read-across approach.

The key study was conducted according to OECD Guideline 429 (LLNA) and is reliable without restrictions (RL1).

The test materials used in the respective studies represent the source substance as described in the hypothesis in terms of substance identity and minor constituents.

Overall, the study results are adequate for the purpose of classification and labelling and risk assessment.

 

Conclusion

Based on structural similarities of the target and source substance as presented above and in more detail in the general justification for read-across, it can be concluded that the available data from the source substance Amphopropionate C8 are also valid for the target substance Amphopropionates C12 -18. This read-across is further supported by the available sensitization study (GPMT) conducted with the target substance itself. Though not fully reliable, the study may suggest a weak sensitization potential.

 

References

Aungst, 1989. Structure/Effect Studies of Fatty Acid Isomers as Skin Penetration Enhancers and Skin Irritants. Pharmaceutical Research, March 1989, Volume 6, Issue 3, pp 244-247

EU RAR, 2002: European Union, Risk Assessment Report: Acrylic acid, CAS No: 79-10-7, Risk Assessment.European Union Risk Assessment Report, 1st Priority List, Vol. 2

 

HERA, 2002: Fatty Acid Salts – Human Health Risk Assessment

 

Ramírez M, Amate L, Gil A. Absorption and distribution of dietary fatty acids from different sources. Early Human Development 2001 Nov;65 Suppl:S95-S101

 

Stillman et al., 1975. Relative irritancy of free fatty acids of different chain length. Contact Dermatitis. 1975;1(2):65-9

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfilled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account.

Justification for classification or non-classification

Based on the available data Amphopropionates C12-18 is classified as Category 1B based on CLP, EU GHS (Regulation (EC) No 1272/2008).