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Diss Factsheets

Administrative data

Description of key information

The in vitro skin irritation study identified as key study was performed according to OECD 439 and under GLP with validity rating 1. The nonylphenol ethoxylate, sarcosine derivative (CAS no 75627-31-5) was tested in the Human Skin Model Test, where tissues of the human skin model EpiDerm™ were treated with substance for 60 minutes. After further incubation for about 42 h the tissues were treated with the MTT solution for 3 h following approximately 72 h extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm. After treatment with the nonylphenol ethoxylate, sarcosine derivative (CAS no 75627-31-5) the mean relative absorbance value was reduced to 5.3% (threshold for irritancy of ≤ 50%). Therefore, the test item is considered to possess an irritant potential. Based on these results and according to Regulation (EC) 1272/2008, the test item has obligatory labelling requirement for skin irritation and has to be classified into Category 2. The Signal Word “Warning” and Hazard Statement “H315: Causes skin irritation” are therefore required. 
The in vivo eye irritation study identified as key study was performed according to OECD 405 and under GLP with validity rating 1. A single application of the test item to the non-irrigated eye of two rabbits produced diffuse to translucent corneal opacity, iridial inflammation and moderate conjunctival irritation. Alopecia around the treated eye was also noted in one animal. The test item produced a maximum group mean score of 34.5 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. Both treated eyes appeared normal at the 14 day observation. Based on the result, the test item was classified as Irritating to eyes (Category 2) according to Regulation (EC) No 1272/2008. The Signal Word “Warning” and Hazard Statement “H319: Causes serious eye irritation” are therefore required.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin 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:
other: According to: OECD Guideline for the testing of Chemicals 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original Guideline adopted July 22, 2010)
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Amount / concentration applied:
Each 35.5 µL of the undiluted test item were applied to three EpiDerm (MatTek) tissues. Due to the purity of 81.7% of the test item, the applied volume was adjusted from 30 µL to 35.5 µL.
Duration of treatment / exposure:
The skin equivalents were exposed to the test item for 60 minutes. After completion of the treatment the test item was rinsed off and the skin equivalents were incubated for further 42 hours.
Details on study design:
Each 35.5 µL of the undiluted test item were applied to three EpiDerm (MatTek) tissues. Due to the purity of 81.7% of the test item, the applied volume was adjusted from 30 µL to 35.5 µL. From the negative control (DPBS) and the positive control (5% SLS) each 30 µL were applied to three tissues. The test item as well as the controls were rinsed off the tissues after 60 minutes treatment. After further 42 hours incubation the tissues were treated with the MTT solution for 3 hours following approximately 72 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at a wavelength of 570 nm.
Irritant / corrosive response data:
After treatment with the of the test item Nonylphenol ethoxylate, sarcosine derivative the mean relative absorbance value was reduced to 5.3% (threshold for irritancy of ≤ 50%). Therefore, the test item is considered to possess an irritant potential.

Results after treatment with Nonylphenol ethoxylate, sarcosine derivative and the controls

 

Dose Group

Treatment Interval

Absorbance 570 nm
Tissue 1*

Absorbance 570 nm
Tissue 2*

Absorbance 570 nm
Tissue 3*

Mean Absorbance of 3 Tissues

%Absorbance

Stand. Deviation

Mean Rel. Absorbance

[% of Negative Control]**

Nega-tive Control

60 min

1.938

1.905

1.899

1.914

101.3

99.5
99.2

1.1

100.0

Positive Control

60min

0.086

0.082

0.090

0.086

5.2

5.4
5.4

0.2

4.5

Test Item

60 min

0.099

0.104

0.103

0.102

4.5

4.3
4.7

0.1

5.3

 

*       Mean of three replicate wells after blank correction
**
      relative absorbance [rounded values]:

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 5.3% (threshold for irritancy:≤50%), consequently the test item was irritant to skin.

Since the present result was not borderline, the performance of the IL-1α Immunoassay was not necessary.

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Nonylphenol ethoxylate, sarcosine derivative is irritant to skin category 2 according to Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008.
Executive summary:

This in vitro study was performed to assess the irritation potential of Nonylphenol ethoxylate, sarcosine derivative by means of the Human Skin Model Test.

Each three tissues of the human skin model EpiDermwere treated with the test item, the negative or the positive control for 60 minutes.

35.5 µL of the undiluted test item were applied to each of triplicate tissues. Due to the purity of 81.7% of the test item, the applied volume was adjusted from 30 µL to 35.5 µL.

30 µL of either the negative control (DPBS) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following approximately 72 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the mean absorbance values were well above therequired acceptability criterion of mean OD greater or equal than 1.0 and less or equal than 2.5 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the mean relative absorbance as compared to the negative control to 4.5% thus ensuring the validity of the test system.

The standard deviations between the % variabilities of the test item, the positive and negative controls were below 2% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

After treatment with the test item Nonylphenol ethoxylate, sarcosine derivative the mean relative absorbance value was reduced to 5.3% (threshold for irritancy of 50%). Therefore, the test item is considered to possess an irritant potential.

Due to the unambiguous result of the test, the performance of the IL-1 α Immunoassay was not necessary.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 03 December 2012 and 03 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.38 or 2.58 kg and were twelve to twenty weeks old. After an acclimatization period of at least five days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye remained untreated and was used for control purposes.
Amount / concentration applied:
A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
14 days
Number of animals or in vitro replicates:
2 animals were tested in total. (After consideration of the ocular responses produced in the first treated animal, one additional animals were treated.)
Details on study design:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Appendix 1.

After consideration of the ocular responses produced in the first treated animal, a second animal was treated.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977) given in Appendix 2.

Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Any clinical signs of toxicity, if present, were also recorded.

An additional observation was made on Day 7 to assess the reversibility of the ocular effects.

Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Irritation parameter:
cornea opacity score
Basis:
animal: 72753 Male
Time point:
other: Highest score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
animal: 72817 Male
Time point:
other: Highest score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
animal: 72753 Male
Time point:
other: Highest score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
animal: 72817 Male
Time point:
other: Highest score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
other: redness
Basis:
animal: 72753 Male
Time point:
other: Highest score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
other: redness
Basis:
animal: 72817 Male
Time point:
other: Highest score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal: 72753 Male
Time point:
other: Highest score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: Alopecia around treated eye
Irritation parameter:
chemosis score
Basis:
animal: 72817 Male
Time point:
other: Highest score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritant / corrosive response data:
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.

Diffuse corneal opacity was noted in both treated eyes one hour after treatment. Translucent corneal opacity was noted in one treated eye with diffuse corneal opacity noted in the other treated eye at the 24, 48 and 72-Hour observations.

Iridial inflammation was noted in both treated eyes one hour after treatment and at the 24, 48 and 72 Hour observations.

Moderate conjunctival irritation was noted in both treated eyes one hour after treatment and at the 24 and 48 Hour observations. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in the other treated eye at the 72 Hour observation. Minimal conjunctival irritation was noted in both treated eyes at the 7 Day observation.

Alopecia around the treated eye was noted in one animal at the 24, 48, 72 Hour and 7 Day observations.

Both treated eyes appeared normal at the 14-Day observation.
Other effects:
Body weight
Individual body weights and body weight changes are given in Table 3.

Both animals showed expected gain in body weight during the study.

Interpretation of Results

The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

 

Score for conjunctivae      =      (A + B + C) x 2

Score for iris                     =      D x 5

Score for cornea               =      (E x F) x 5

 

Using the numerical data obtained a modified version of the system described by Kay J.H. and Calandra J.C. (1962)(see Appendix 3) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test item.

 

If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.

 

The results were also interpreted according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Table 1     Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

72753Male

72817Male

IPR= 2

IPR = 2

Time After Treatment

1
Hour

24
Hours

48
Hours

72
Hours

7
Days

14
Days

1
Hour

24
Hours

48
Hours

72
Hours

7
Days

14
Days

CORNEA

 

 

 

 

 

 

 

 

 

 

 

 

E = Degree of Opacity

1

2

2

2

0

0

1

1

1

1

0

0

F = Area of Cornea Involved

2

3

3

2

0

0

1

1

1

1

0

0

Score (E x F) x 5

10

30

30

20

0

0

5

5

5

5

0

0

IRIS

 

 

 

 

 

 

 

 

 

 

 

 

D

1

1

1

1

0

0

1

1

1

1

0

0

Score (D x 5)

5

5

5

5

0

0

5

5

5

5

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

 

 

 

A = Redness

2

2

2

2

1

0

2

2

1

1

1

0

B = Chemosis

2

2

2

2

1

0

2

2

1

1

1

0

C = Discharge

3

1Al

1Al

1Al

0Al

0

3

3

2

1

0

0

Score (A + B + C) x 2

14

10

10

10

4

0

14

14

8

6

4

0

Total Score

29

45

45

35

4

0

24

24

18

16

4

0


IPR=Initial pain reaction

Al =        Alopecia around treated eye

Table 2     Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number

and Sex

Individual Total Scores At:

1 Hour

24 Hours

48 Hours

72 Hours

7 Days

14 Days

72753Male

29

45

45

35

4

0

72817Male

24

24

18

16

4

0

Group Total

53

69

63

51

8

0

Group Mean Score

26.5

34.5

31.5

25.5

4.0

0.0

Table 3     Individual Body Weights and Body Weight Change

Rabbit Number
and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

Day 0

Day 14

72753Male

2.38

2.66

0.28

72817Male

2.58

2.97

0.39

Interpretation of results:
other: The test item produced a maximum group mean score of 34.5 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale)
Remarks:
Criteria used for interpretation of results: other: modified Kay and Calandra classification system
Conclusions:
The test item produced a maximum group mean score of 34.5 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item was also classified as Irritating to eyes (Category 2) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. The Signal Word “Warning” and Hazard Statement “H319: Causes serious eye irritation” are therefore required.
Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

 

 

Results

A single application of the test item to the non-irrigated eye of two rabbits produced diffuse to translucent corneal opacity, iridial inflammation and moderate conjunctival irritation. Alopecia around the treated eye was also noted in one animal. Both treated eyes appeared normal at the 14‑Day observation.

 

 

Conclusion

The test item produced a maximum group mean score of 34.5 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

 

The test item was also classified as Irritating to eyes (Category 2) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. The Signal Word “Warning” and Hazard Statement “H319: Causes serious eye irritation” are therefore required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin
Three skin irritation studies of different quality and different methods are availableonnonylphenol ethoxylate, sarcosine derivative (CAS no 75627-31 -5). The key study is an in vitro study performedaccording to OECD 439 and under GLP conditions. In this study tissues of the human skin model EpiDermwere treated with the test item, the negative or the positive control for 60 minutes. The result from this study indicates that the substance is irritating to skin Cat 2. The supporting study is an older in vivo study performed according to OECD 404 on a formulation containingnonylphenol ethoxylate, sarcosine derivative (CAS no 75627-31-5). The result from this study indicated the tested formulation to have skin Cat 3, which is in line with the key study since a skin Cat 2 substance may generate skin Cat 3 if diluted. The third study, which is an in vitro skin corrosion test, does also support the classification of the substance as non-corrosive, but it cannot evaluate the irritation potential and therefore the key study was performed.   


Eye 

Two eye irritation studies are available on nonylphenol ethoxylate, sarcosine derivative (CAS no 75627-31-5), but they give conflicting results. It is one in vitro BCOP study and one in vivo study, both performed under GLP and according to guidelines. The prevailing in vivo study is chosen as key study and it shows that the substance is irritating to eyes although the in vitro study BCOP study gives no such indications.


Justification for selection of skin irritation / corrosion endpoint:
The chosen study is an in vitro Klimisch 1 GLP skin irritation study performed on Nonylphenol ethoxylate, sarcosine derivative (CAS no 75627-31-5) according to the current OECD guideline 439.

Justification for selection of eye irritation endpoint:
The chosen study is an in vivo Klimisch 1 GLP eye irritation study performed on Nonylphenol ethoxylate, sarcosine derivative (CAS no 75627-31-5) according to the current OECD guideline 405.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin

The conclusion from the three available studies is that the substancenonylphenol ethoxylate, sarcosine derivative (CAS no 75627-31-5) is not corrosive buthas to be classified for skin irritation as Category 2 according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

 

Eye

The in vivo eye irritation study is the prevailing study and it gives GHS Cat 2 for eye irritation, although no irritation is seen in the in vitro BCOP study.Nonylphenol ethoxylate, sarcosine derivative (CAS no 75627-31-5) is to be classified for eye irritation Category 2 according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.