Amines, C12-16 alkyl dimethyl, reaction products with ethyl chloroacetate, 2-(2-aminoethylamino)ethanol and 2-propenoic acid

Endpoint summary

Description of key information

Skin irritation:  not irritating
Eye irritation: corrosive
Under the conditions of this study the test substance was graded as moderately irritating to eyes and mucous membranes according to the Primary Irritation Index. However, Rewoteric V 0814 (10 % solution) has irreversible effects on the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion, other

Remarks:

The skin irritation potential was assessed by rthe results obtained in a dermal toxicity study.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 June 2015 - 23 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

other: CD / Crl: CD(SD)

Details on test animals or test system and environmental conditions:

Strain / Stock CD / Crl: CD(SD)
Supplier: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Body weight (at dosing):Males: 213 - 223 g; Females: 206 - 234 g
Age (at dosing): Males: approx. 8 weeks; Females: approx. 9 weeks
Identification of animals: By coloured marks and cage label

Commercial diet, ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. Feeding was discontinued approx. 16 hours before administration; only tap water was then available ad libitum.
Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL. Certificates of analysis of the composition and for contaminants were provided by the manufacturer and are included in the raw data.
Housing
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned twice a week.
Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL (see Appendix 2 'Limitation for Contaminants in the Bedding Material').
During the 14-day observation period the animals were kept singly in MAKROLON cages (type III plus) at a room temperature of 22°C ± 3°C (maximum range) and a relative humidity of 55% ± 15% (maximum range). Deviations from the maximum range caused for example during cleaning procedures are dealt with in SOPs.
The rooms were lit (150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each.

Drinking water
Drinking water in bottles was offered ad libitum.
Drinking water is examined according to the 'Deutsche Trinkwasserverordnung 2011 [German Regulations on drinking water 2011] by the Hamburger Wasserwerke, 20539 Hamburg, Germany, at least four times a year (see Appendix 2 'Limitation for Contaminants in the Drinking Water').
In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2011, Anlage 1' [German Regulations on drinking water 2011, Addendum 1].

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

The test substance was used as supplied. The application volume was 3.73 mL/kg b.w. as the density of the test item was 1.0731 g/mL.
The test item was an approx. 50% aqueous solution. A correction factor of 2 was employed. The concentration refers to the solid content (active ingredient).

Duration of treatment / exposure:

24 hours

Observation period:

2 weeks

Number of animals:

5 animals/sex

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 5, 15, 30 min after administration, as well as 3, 6 and 24 hours after administration
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology: changes of skin and fur, eyes and mucous membranes, respiratory and circulatory function, autonomic and central nervous system and somatomotor activity as well as behaviour pattern, were observed at least once a day until all symptoms subsided, thereafter each working day. Attention was also paid to possible tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Observations on mortality were made at least once daily to minimize loss of animals during the study. Individual body weights were recorded before administration of the test item and thereafter in weekly intervals up to the end of the study. Changes in weight were calculated and recorded.
The skin was observed for the development of erythema and oedema.

Irritation parameter:

other: visual inspection

Basis:

other: all animals in test

Time point:

other: daily until end of test

Remarks on result:

no indication of irritation

Summarized results

Symptoms/Criteria	Test substance 2000mg/kg b.w. (n = 5)
	males	females
Clinical signs	none	none
Skin reactions	none	none
Mortality within 6 h within 27 h within 7 d within 14 d	0 0 0 0	0 0 0 0
Mean body weight (in g) start	217.6	218.2
After 7 days	275.8 (+ 26.7)	238.6 (+ 9.3)
After 14 days	331.8 (+ 52.5)	263.8 (+ 20.9)
Inhibition of body weight gain	none	None
Necropsy findings	none	none

 

In brackets: body weight gain in %, compared to the start value

The dose level refers to the active ingredient

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

No skin reactions were observed at the application site 24 hours after application until end of observation period (2 weeks).

Executive summary:

There is no evidence for a skin irritating potential of Amines, C12-16 alkyl dimethyl, reaction products with ethyl chloroacetate, 2-(2-aminoethylamino)ethanol and 2-propenoic acid from an acute dermal toxicity study (OECD guideline 402). Groups of 5 male and female young adult rats (Rattus norvegicus) were dermally exposed to the test substance for 24 hours under an occlusive dressing. The applied dose of 2000 mg/kg bw (related to a.i.) is equivalent to an average concentration of 29.1 mg/cm² and 29.4 mg/cm² for males and females, respectively. Calculation is based on mean applied volume males: 0.81 mL, females: 0.82 mL; densitiy 1.0731 g/mL and area of exposue 5 x 6 cm.

No signs of irritation were recorded during the 14 day observation period.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October/November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

26 July 2012 Date of Signature: 11 April 2013

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

a volume of 0.75 mL of the undiluted test item was applied on the surface of the bovine corneae.

Duration of treatment / exposure:

The incubation time lasted 10 minutes.

After the test item or control items, respectively, were rinsed off from the application side with saline, the corneae were incubated for further two hours in a vertical position.

Observation period (in vivo):

approx. 130 minutes in total

Number of animals or in vitro replicates:

3 corneae for the test item plus 3 corneae for the negative and positive control, respectively.

Details on study design:

This test is designed to measure the opacity of the cornea by quantifying the ability of light to pass through it. The permeability, as a result of the irritation potential of the test item, was determined using Na-fluorescein solution. The comparison of the opacity before and after the exposure to the test item and the determination of the permeability after the treatment provide an indication of the damaging effect of the test item.
For this purpose the induction of opacity and increased permeability in an isolated bovine cornea after application of the test item were measured.

2-Ethoxyethanol served as positive control. Saline was used as negative control.

Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
For equilibration and prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).


Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer (software SoftMax Pro Enterprise, version 4.7.1).

DATA INTERPRETATION

Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:
IVIS In vitro Irritancy Score (according to OECD 437)
≤ 3 No Category (according to GHS)
> 3; < 55 No prediction can be made
≥ 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)

Irritation parameter:

in vitro irritation score

Value:

99.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

Relative to the negative control, the test item Rewoteric QAM 50 caused a strong increase of the corneal opacity and the permeability (both higher than the ones caused by the positive control).

Results after 10 Minutes Incubation Time

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	0	0	0.050	0.051	0.75	0.76	Not categorized
	0		0.046		0.69
	0		0.056		0.84
Positive Control	54.00		0.636*		63.55	64.42	Category 1
	50.00		0.911*		63.67
	50.00		1.069*		66.04
Test substance	83.00		1.308*		102.63	99.33	Category 1
	70.00		1.608*		94.13
	83.00		1.216*		101.25

* corrected values

 

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

Migrated information

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, Rewoteric QAM 50 is seriously eye damaging.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of the substance by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae.

Relative to the negative control, the test item caused a strong increase of the corneal opacity and the permeability. The calculated mean IVIS was 99.33 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 the test item is classified as seriously eye damaging.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substance was tested in 5 % concentration regarding its skin irritation potential by occluded application of the test substance for 24 hours to scarified and intakt skin of six male rabbit. Only slight skin reactions were observed 24 h after test substance removal which were fully reversible until 72 h. From the test data it is concluded that the 5 % test substance is not irritating to skin.

To assess the corrosive potential of the substance a Human Skin Model Test was performed. The test item was not considered to be corrosive.

In an OECD 402 acute dermal toxicity test no skin reactions were observed in all animals 24 hours after treatment until end of observation period (two weeks).

In a BCOP assay using fresh bovine corneae the substance caused strong effects on the corneal opacity and the permeability. Under the experimental conditions reported the substance is seriously eye damaging and should be classified to Category 1 according to CLP/EPA/GHS.

Justification for selection of skin irritation / corrosion endpoint:
Data from an acceptable, well-documented study report with reliability 1.

Justification for selection of eye irritation endpoint:
Data from a GLP Guideline study report with reliability 1.

Effects on eye irritation: corrosive

Justification for classification or non-classification

Skin irritation

Based on reliable, adequate and relevant data, the substance does not need to be classified for skin irritation.

 

Eye irritation

Based on reliable, adequate and relevant data, the substance needs to be classified in Category 1, irreversible effects on the eye according to CLP, EU GHS (Regulation (EC) No 1272/2008).