Amines, C12-14-alkyl, C6-10-alkyl phosphates

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Deviations:

The following deviations from the study plan were observed:
• The pre-test for the ability of direct formazan reduction was not incubated at room
temperature but in the incubation chamber at 3rC and 5% C02. The deviation can
be seen as uncritical since the SOP of MatTek and the SOP of the LAUS GmbH (118
00 820) prescribes it this way (error in study plan).
The deviationwas assessed and signed by the deputy study director on 5. Nov. 2010.
• lnstead of the analytical scales Mettier Toledo AB 184 SA, the analytical scales Mettier
Toledo XS 205 DU were used. The deviation can be seen as uncritical since the
new analytical scales work as precisely as the old analytical scales.
The deviationwas assessed and signed by the deputy study director on 9. Dec. 2010.

Deviations:

yes

Remarks:

The deviation can be seen as uncritical

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

Commercially available Epi-200-Kit. The EpiDerm TM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. lt consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar Iipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The liquid test item was applied without preparation (50 µL).

Duration of treatment / exposure:

three minutes and one hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

ca. 114.4

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

ca. 113.4

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item is considered not corrosive. After three minutes treatment, the relative absorbance values were increased to 114.4%. This value is well above the threshold for corrosivity (50%). After one hour treatment relative absorbance values were increased to 113.4%. This value is well below the threshold for corrosivity (15%). The values of the negative control were well above the required acceptability criterion of mean OD>O.8 for both treatment interval thus showing the quality of the tissues. The value of the positive control induced a decrease in the relative absorbance as compared to the negative control to 30.5% for the three minutes treatment interval and 12.9% for the one hour treatment interval thus ensuring the validity of the test system. For these reasons, the result of the test is considered valid.

Executive summary:

One valid experimentwas performed. Two tissues of the human skin model EpiDerm TM were treated with the test substance for three minutes and one hour, respectively. 50 µL of the liquid test item were applied to each tissue and spread to match the tissue size. Deionised water was used as negative control, 8 m KOH was used as positive control. After treatment with the negative control the absorbance values were weil above the required acceptability criterion of mean OD > 0.8 for both treatment intervals thus showing the quality of the tissues. The positive control showed clear corrosive effects for both treatment intervals. After three minutes treatment with the test item, the relative absorbance values were increased to 114.4 %. This value is weil above the threshold for corrosion potential (50%). After one hour treatment, relative absorbance values were increased to 113.4 %. This value, too, is weil below the threshold for corrosion potential (15%). Therefore, the test substance is considered as not corrosive in the Human Skin Model Test.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 f..JL test item were applied

Duration of treatment / exposure:

Tissues were dosed in one minute intervals. After 24 minutes, all plates are transferred into the incubator for 35 min.. 60 min. after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in one-minute-intervals.

Duration of post-treatment incubation (if applicable):

18 ± 2 hours for post-incubation

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

ca. 10.6

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item is considered irritant. Study No.: 1 0092802G840. Test ltem: Deophos 228. After the treatment, the relative absorbance values were decreased to 10.6%. This value is weil below the threshold for irritation (50%). The optical density of the negative control was weil within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control induced a decrease in the relative absorbance as compared to the negative control to 7.0% (required: ~ 20%) for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item. For these reasons, the result of the test is considered valid.

Executive summary:

One valid experiment was performed. Three tissues of the human skin model EpiDerm TM were treated with the test substance for 60 minutes. 30 1-JL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size. DPBS-buffer was used as negative control, 5% SOS-solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18%). After the treatment with the test item, the relative absorbance values were reduced to 10.6 %. This value is weil below the threshold for irritation potential (50%). Therefore, the test substance 228 is considered as irritant in the Human Skin Model Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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)

Version / remarks:

Deviations:

The following deviations from the study plan were observed:
+ Cuvettes with a pathlength of 0.2 cm were used in the measurement of the Fluorescein-
Na solution in the spectrophotometer and all absorptions were multiplied with
the coefficient 5. The deviation was considered as uncritical since the test results
were not influenced.
+ The positive control benzalkonium chloride solution (5%) was dissolved in demineralised
water instead of 0.9% sodium chloride solution. The deviation can be seen as
uncritical since it was incorrectly stated in the study plan.
The deviations were assessed and signed by the deputy study director on 11. Jan. 2011.

Deviations:

yes

Remarks:

The deviation can be seen as uncritical

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Freshly bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank's balanced salt solution (supplemented with 0.01% Streptomycin and 0.01% penicillin). Then the corneas were dissected and incubated with media at 32 ± 1 oc in an incubation chamber for 1 hour.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

in average, 214.3 mg of the test item were tested neat and applied directly on the cornea using a weight board. The test item was given on the epithelium in such a manner that as much as possible of the corneawas covered with test item. The test item is a surface active liquid. Surfactants (solids or liquids) are usually tested at a concentration of 10% in a 0.9% sodium chloride solution, deionised water or other solvents which have no adverse effects in the test system. The test item Deophos 228 was not soluble in one of the solvents mentioned above in the demanded concentration (10%). Since no solutionwas feasible, the test item was applied directly on the cornea without dilution or preparation of a solution.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

90 ± 5 min

Number of animals or in vitro replicates:

3

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

ca. 0.785

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

IVIS was calculated using the values in tables 9.1-a and 9. I-b and the equation stated in chapter 8.3. Example: IVIS (Deophos 228, Repl. 1) = (0.0893 - 0.2416) + [15 * (5 * 0.0142 - 0.0200)] = 0.6127

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Table 9.2.a IVIS

			Relative Standard Deviation IVIS
Negative Control 0,9% NaCl	0.6757	0.5420	21.4%
	0.4836
	0.4671
Deophos 228	0.6127	0.7850
	1 .5786
	O. 1644
Positive Control 5% Benzalkonium chloride solution	88.5716	108.6360	17.0%
	112.4971
	124.8378

Note: the high relative standard deviation of the IVIS of test item is due to mathematical reasons, as the respective mean is very small.

Parameter                                               Criterion

IVIS of ne ative control 0.9% NaCl	0 - 3	Found 0.5420	Assessment ok
IVIS of positive control 5% Benzalkonium chloride solution	55.4 —	108.6360	Too high

Values for negative control were within the range of historical data of the test facility (see 14, page 20). Therefore, the test system was acceptable. The values for positive control were not within the range of historical data of the test facility. This can be seen as uncritical because the range of the historical data was quite narrow since it referred only to six individual replicates. Besides that, the high value of the positive control can be seen as uncritical because of the fact that it showed clearly very severe eye irritating effects which confirms that the test system is acceptable.

Interpretation of results:

GHS criteria not met

Conclusions:

This in vitro study was performed to assess the corneal irritation and darnage potential of Deophos 228 by quantitative measurements of changes in opacity and permeability in a bovine cornea. The test item Deophos 228 was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without Phenol red at 32 ± 1°C for one hour and whose opacity had been determined. The lest item was incubated on the cornea for 10 minutes at 32 ± 1°C. After removal of the lest item and two hours post-incubation, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control; benzalkonium chloride (5% solution in demineralised water) was used as positive control. The positive control induced a very severe irritation on the cornea, mean IVIS was 108.6360. The negative control showed no irritation, mean IVIS was 0.5420. The lest item was tested pure. A mean IVIS of 0.7850 was calculated, corresponding to a classification as not eye irritant. No observations were made which might cause doubts concerning the validity of the study outcome. The lest is considered valid.

Executive summary:

One valid experiment was performed. Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old. The test item was brought onto the cornea of a bovine eye which had been incubated with cMEM without Phenol red at 32 ± 1°C for one hour and whose opacity had been measured. The test item was incubated on the cornea for 10 minutes at 32 ± 1°C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the cornea. 5% benzalkonium chloride solution was used as positive control. The positive control induced a very severe irritation on the cornea. The test item showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 0. 7850. ln conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses no eye irritation potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

One valid experimentwas performed. Two tissues of the human skin model EpiDerm TM were treated with the test substance for three minutes and one hour, respectively. 50 µL of the liquid test item were applied to each tissue and spread to match the tissue size. Deionised water was used as negative control, 8 m KOH was used as positive control. After treatment with the negative control the absorbance values were weil above the required acceptability criterion of mean OD > 0.8 for both treatment intervals thus showing the quality of the tissues. The positive control showed clear corrosive effects for both treatment intervals. After three minutes treatment with the test item, the relative absorbance values were increased to 114.4 %. This value is weil above the threshold for corrosion potential (50%). After one hour treatment, relative absorbance values were increased to 113.4 %. This value, too, is weil below the threshold for corrosion potential (15%). Therefore, the test substance is considered as not corrosive in the Human Skin Model Test.

One valid experiment was performed. Three tissues of the human skin model EpiDerm TM were treated with the test substance for 60 minutes. 30 1-JL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size. DPBS-buffer was used as negative control, 5% SOS-solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18%). After the treatment with the test item, the relative absorbance values were reduced to 10.6 %. This value is weil below the threshold for irritation potential (50%). Therefore, the test substance 228 is considered as irritant in the Human Skin Model Test.

One valid experiment was performed. Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old. The test item was brought onto the cornea of a bovine eye which had been incubated with cMEM without Phenol red at 32 ± 1°C for one hour and whose opacity had been measured. The test item was incubated on the cornea for 10 minutes at 32 ± 1°C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the cornea. 5% benzalkonium chloride solution was used as positive control. The positive control induced a very severe irritation on the cornea. The test item showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 0. 7850. ln conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses no eye irritation potential.

Justification for classification or non-classification

Skin irritation:

Based on the results of the in vitro skin irritation studies, the substance is considered to be irritant to skin and hence warrants a classification as Skin Irrit. 2 ( H315: Causes skin irritation) according to EU CLP (1272/2008/EC) criteria.

Eye irritation:

Considering the results of the in vitro eye irritation study, the substance does not warrant any classification for eye irritation according to EU CLP (1272/2008/EC) criteria.