Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate

Endpoint summary

Description of key information

The test material was considered to be a non sensitiser under the conditions of the test (LLNA), OECD 429, EU Method B.42, Bradshaw (2009).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Justification for type of information:

An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 05 February 2009 and 18 February 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007

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: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Limited, Bicester, Oxon, UK and female CBA/Ca (CBA/CaBkl) strain mice were supplied by B & K Universal Ltd, Hull, UK.
- Age at study initiation: eight to twelve weeks old.
- Weight at study initiation: At the start of the study the animals were in the weight range of 15 to 23 g
- Housing: The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet : Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study. ad libitum
- Water: Free access to mains tap water ( ad libitum)
- Acclimation period: acclimatisation period of at least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): in the range 19 to 25°C
- Humidity (%): in the range 30 to 70%,
- Air changes (per hr): The rate of air exchange was approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.


IN-LIFE DATES: From: Day 0 To: Day 6

Vehicle:

other: 1% pluronic L92 in distilled water,

Concentration:

10%, 5% or 2.5% w/w in 1% pluronic L92 in distilled water.

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Not stated in report
- Irritation: None
- Lymph node proliferation response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC
- Criteria used to consider a positive response:
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:

The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.
3H-Methyl Thymidine Administration:
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.

Positive control substance(s):

other: 2,4-Dinitrobenzenesulfonic acid, sodium salt

Statistics:

Not stated in report

Positive control results:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:
Concentration (% w/w) in
1% pluronic L92 in distilled water Stimulation Index Result
10 13.71 Positive

Conclusion: 2,4 Dinitrobenzenesulfonic acid, sodium salt was considered to be a sensitiser under the conditions of the test.

Parameter:

SI

Value:

1.32

Test group / Remarks:

2.5 % w/w test material in 1 % pluronic L92 in distilled water

Parameter:

SI

Value:

1.13

Test group / Remarks:

5 % w/w test material in 1 % pluronic L92 in distilled water

Parameter:

SI

Value:

1.15

Test group / Remarks:

10 % w/w test material in 1 % pluronic L92 in distilled water

Parameter:

SI

Remarks on result:

other: Please see table 1 in attachment 1

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: please see table 1 in attachement 1

 Preliminary Screening Test:

Clinical observations, bodyweight and mortality data are given below:

Concentration (%w/w) in 1% pluronic L92in distilled water	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
10	S-1	17	17	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% w/w in 1% pluronic L92 in distilled water.

Main Test:

Estimation of the Proliferative Response of Lymph Node Cells:

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1 of attachment 1.

A stimulation index of less than 3 was recorded for the three concentrations of the test material (10%, 5% and 2.5% w/win 1% pluronic L92 in distilled water).

Clinical Observations and Mortality Data:

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight:

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period except for one animal treated with the test material at a concentration of 5% w/w in1 % pluronic L92 in distilled water which showed a slightly higher than expected bodyweight loss.

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

The test material was considered to be a non sensitiser under the conditions of the test.

Executive summary:

Introduction:

A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

-   OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

-      Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC.

Methods:

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test material as a solution in 1% pluronic L92 in distilled water at concentrations of 10%, 5% or 2.5% w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water alone.

Results: 

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in 1% pluronic L92 in distilled water	Stimulation Index	Result
2.5	1.32	Negative
5	1.13	Negative
10	1.15	Negative

Conclusion:

The test material was considered to be a non‑sensitiser under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The key study (Bradshaw 2009) was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was conducted in accordance with the standardised guidelines OECD 429 and EU Method B.42 under GLP conditions. The study was therefore assigned a reliability score of 1 in accordance with the criteria for assessing data quality as described in Klimisch et al. (1997).

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a solution in 1 % pluronic L92 in distilled water at concentrations of 10, 5 or 2.5 % w/w. A further group of four animals was treated with 1 % pluronic L92 in distilled water alone.

The Stimulation Index, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, was 1.32, 1.13 and 1.15 at 2.5, 5 and 10 % w/w. these values all indicate a negative result. 

The test material was considered to be a non-sensitiser under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to skin sensitisation.