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Description of key information

The test substance was corrosive to rabbit skin after a 4 hour occluded exposure. The BCOP showed a score below 55 which does not indicate a category 1 classification. However the BCOP test can under predict eye damage for diamine chemistry.An in-vivo rabbit eye study was not possible due to animal welfare concerns as the test substance was corrosive to skin category 1C.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 14 March 2017 Experimental completion date 28 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), Testing Guidelines for Toxicology Studies, 12 NohSan No. 8147, amended 10 December 2002
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Diamine-Fatty Acids mixture salt
Batch: HJ16-040
Purity: 99.6%
Physical state / Appearance: brown paste
Expiry date: 07 July 2017
Storage conditions: room temperature in the dark
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
One New Zealand White (Hsdlf:NZW) strain rabbit was supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animal weighed 3.23 kg and was 12 to 52 weeks old. After an acclimatization period of at least 5 days the 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.

Animal Care and Husbandry
The animal was housed in a suspended cage. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, 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. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.

The animal was 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.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
On the day before the test the rabbit was clipped free of fur from the dorsal/flank area using veterinary clippers.
Three suitable sites were selected on the back of the rabbit. A quantity of 0.5 mL of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. Each patch was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset for the duration of the exposure period.
Duration of treatment / exposure:
One patch was removed at each of three time points: 3 minutes, 1 hour and 4 hours after application. Any residual test item was removed by gentle swabbing with cotton wool soaked in 74% Industrial Methylated Spirits.
After consideration of the skin reactions produced in the first animal, no additional animals were treated.
Observation period:
14 days
Number of animals:
One
Details on study design:
One patch was removed at each of three time points: 3 minutes, 1 hour and 4 hours after application. Any residual test item was removed by gentle swabbing with cotton wool soaked in 74% Industrial Methylated Spirits.

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test item is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable.
The results were also evaluated according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures and the Globally Harmonized System of Classification and Labelling of Chemicals.


Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.33
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
3-Minute Exposure Period
Well-defined erythema and very slight edema were noted at the treated skin site at the 24 Hour observation with well defined erythema and slight edema noted at the 48 Hour observation and well defined erythema and very slight edema noted at the 72 Hour observation. Slight desquamation was noted at the treated skin site at the 7 Day observation.

The treated skin site appeared normal at the 14-Day observation.

1-Hour Exposure Period
Very slight erythema and very slight edema were noted at the treated skin site immediately and 1 hour after treatment. Well defined erythema and slight edema were noted at the treated skin site at the 24 Hour observation with well defined erythema and very slight edema noted at the 48 and 72 Hour observations. The erythema and edema extended approximately 10 mm around the test site at the 24, 48 and 72 Hour observations.

Additional skin reactions noted at the treated skin site at the 24, 48 and 72 Hour observations were hemorrhage of the dermal capillaries, blanching of the skin, light brown discoloration of the epidermis and loss of skin elasticity and flexibility. A hardened dark brown/black colored scab, preventing accurate evaluation of erythema and edema, was noted at the treated skin site at the 7 Day observation. Small superficial scattered scabs and glossy skin were noted at the treated skin site at the 14 Day observation.

4-Hour Exposure Period
Very slight erythema and slight edema were noted at the treated skin site immediately after patch removal with well defined erythema and slight edema noted 1 hour after treatment. Well defined erythema and moderate edema were noted at the treated skin site at the 24 Hour observation with well defined erythema and slight edema noted at the 48 and 72 Hour observations. The erythema and edema extended approximately 10 mm around the test site at the 24 Hour observation.

Additional skin reactions noted at the treated skin site at the 24, 48 and 72 Hour observations were hemorrhage of the dermal capillaries, blanching of the skin, light brown discoloration of the epidermis and loss of skin elasticity and flexibility. A hardened dark brown/black colored scab, preventing accurate evaluation of erythema and edema, was noted at the treated skin site at the 7 Day observation. Small superficial scattered scabs, scar tissue and glossy skin were noted at the treated skin site at the 14 Day observation.

The skin reactions noted were considered to be irreversible and indicative of full thickness tissue destruction.
Other effects:
The animal showed expected gain in body weight during the study.

Individual Skin Reactions Following 3-Minute and 1-Hour Exposures

Skin Reaction

Observation Time
(following patch removal)

Individual Scores

Rabbit Number and Sex

75729Female

3-Minute Exposure

1-Hour Exposure

Erythema/Eschar Formation

Immediately

0

1

1 Hour

0

1

24 Hours

2

2RHdBlLeLfBr

48 Hours

2

2RHdBlLeLfBr

72 Hours

2

2RHdBlLeLfBr

7 Days

0D

?eSt

14 Days

0

0SsG

Edema Formation

Immediately

0

1

1 Hour

0

1

24 Hours

1

2

48 Hours

2

1

72 Hours

1

1

7 Days

0

?od

14 Days

0

0

 

Individual Skin Reactions Following 4-Hour Exposure

Skin Reaction

Observation Time
(following patch removal)

Individual Scores

Rabbit Number and Sex

75729Female

Erythema/
Eschar Formation

Immediately

1

1 Hour

2

24 Hours

2RHdBlLeLfBr

48 Hours

2HdBlLeLfBr

72 Hours

2HdBlLeLfBr

7 Days

?eSt

14 Days

0SsScG

Edema Formation

Immediately

2

1 Hour

2

24 Hours

3

48 Hours

2

72 Hours

2

7 Days

?od

14 Days

0

Classification                                                      :              CORROSIVE

 

Individual body Weights and Body Weight Change 

Rabbit Number
and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

Day 0

Day 14

75729Female

3.23

3.51

0.28

 


Bl =                Blanching of the skin

Br =       Light brown discoloration of the epidermis

D=          Slight desquamation

G =         Glossy skin

Hd =                      Hemorrhage of dermal capillaries

Le =       Loss of skin elasticity

Lf =       Loss of skin flexibility

R =         Erythema and edema extended approximately 10 mm around the test site

Ss =      Small superficial scattered scabs

St =        Hardened dark brown/black colored scab

?e =        Adverse skin reaction prevented accurate evaluation of erythema

?od =     Adverse skin reaction prevented accurate evaluation of edema

Bl =                Blanching of the skin

Br =       Light brown discoloration of the epidermis

G =         Glossy skin

Hd =                      Hemorrhage of dermal capillaries

Le =       Loss of skin elasticity

Lf =       Loss of skin flexibility

R =         Erythema and edema extended approximately 10 mm around the test site

Sc =       Scar tissue

Ss =      Small superficial scattered scabs

St =        Hardened dark brown/black colored scab

?e =        Adverse skin reaction prevented accurate evaluation of erythema

?od =     Adverse skin reaction prevented accurate evaluation of edema

Interpretation of results:
Category 1C (corrosive) based on GHS criteria
Conclusions:
The test item was classified as CORROSIVE to rabbit skin (based on one animal only).

The test item was also classified as Category 1C (Corrosive) according to the Globally Harmonized System of Classification and Labeling of Chemicals.

The test item was also classified as Corrosive (Category 1C) according to Regulation (EC) No 1272/2008, relating to the Classification, Labeling and Packaging of Substances and Mixtures. It is reasonable to assume that the signal word “Danger” and the Hazard Statement “H314: Causes severe skin burns and eye damage” are therefore required.
Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item following single, 3‑Minute, 1 and 4‑Hour, semi‑occluded applications to the intact rabbit skin.

Results

A single 3‑Minute semi-occluded application of the test item to the intact skin of one rabbit produced well-defined erythema, slight edema and slight desquamation. The treated skin site appeared normal at the 14‑Day observation.

A single 1‑Hour semi-occluded application of the test item to the intact skin of one rabbit produced well‑defined erythema, slight edema, hemorrhage of the dermal capillaries, blanching of the skin, light brown discoloration of the epidermis, loss of skin elasticity and flexibility, small superficial scattered scabs, a hardened dark brown/black colored scab (preventing evaluation of erythema and edema) and glossy skin. On occasions the erythematous and edematous reactions extended approximately 10 mm beyond the treatment site.

A single 4‑Hour, semi occluded application of the test item to the intact skin of one rabbit produced reactions indicative of dermal corrosion. The reactions included well‑defined erythema, moderate edema, hemorrhage of the dermal capillaries, blanching of the skin, light brown discoloration of the epidermis, loss of skin elasticity and flexibility, a hardened dark brown/black colored scab (preventing evaluation of erythema and edema), small superficial scattered scabs, scar tissue and glossy skin. On occasions the erythematous and edematous reactions extended approximately 10 mm beyond the treatment site.The skin reactions noted were considered to be irreversible and indicative of full thickness tissue destruction.

Conclusion

The test item was classified as CORROSIVE to rabbit skin (based on one animal only).

The test item was also classified as Category 1C (Corrosive) according to the Globally Harmonized System of Classification and Labeling of Chemicals.

The test item was also classified as Corrosive (Category 1C) according to Regulation (EC) No 1272/2008, relating to the Classification, Labeling and Packaging of Substances and Mixtures. It is reasonable to assume that the signal word “Danger” and the Hazard Statement “H314: Causes severe skin burns and eye damage” are therefore required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The test was performed on 04 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 25 April 2012, 23 ,25,Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015
Species:
other: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
0.75 mL of 20% (w/v) suspension of the test item in saline was prepared using ultrasonic technique (5 minutes).
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
The incubation period was 240 minutes.
Number of animals or in vitro replicates:
3 corneae per group (test item, negative control, positive control)
Details on study design:
Three corneas were exposed to each 0.75 mL of the test item, the negative, and the positive control for 10 minutes.

SCORING SYSTEM:
Opacity Measurement:
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. 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.
After exposure of the corneae to the test groups and after rinsing, the opacity value was determined again (t240).

Permeability Determination:
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation 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 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Evaluation

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 (t240 – 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 in 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 CSerious eye damage according to CLP/EPA/GHS (category 1)


Criteria for Determination of a Valid Test:

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of three cornea
Value:
40.21
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No prediction can be made
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Results after 240 Minutes Incubation Time

 Test Group  Opacity value = Difference (t240-t0) of Opacity    Permeability at 490 nm (OD490)    IVIS  Mean IVIS  Proposed in vitro Irritancy Score
     Mean    Mean      
   0    0.087    1.31    
 Negative Control 1  0.33  0.077  0.083  2.16  1.58  Not categorized
   0    0.085    1.28    
 112.67*    0.105*    114.24    
  Positive Control  113.67*    0.184*    116.43  113.77  Category 1
   107.67*    0.199*    110.65    
   21.67*    0.975*    36.29    
 Diamine-Fatty Acids mixture salt  14.67*    1.900*    43.17  40.21  No prediction
   11.67*    1.966*    41.16    

*corrected values

Interpretation of results:
other: Diamine-Fatty Acids mixture salt is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, Diamine-Fatty Acids mixture salt is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).
Executive summary:

This in vitro study was performed to assess thecorneal damage potentialofDiamine-Fatty Acids mixture salt by means of the BCOP assay usingfresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v)suspensionin saline (0.9% (w/v) NaCl in deionised water) of the test item Diamine-Fatty Acids mixture salt, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber containedincubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae andopacity was measured again (t240).

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 (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item Diamine-Fatty Acids mixture salt caused an increase of the corneal opacity and permeability compared with the values caused by the negative control. The calculated meanin vitroirritancy score was 40.21.According to OECD 437 the test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1), but the test item’s hazard for eye damaging cannot be predicted.

Endpoint:
eye irritation: in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study need not be conducted because the available information indicates that the criteria are met for classification as corrosive to the skin or irritating to eyes
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The test substance was corrosive to rabbit skin after 4 hour exposure. Based on this it is classified as category 1C for corrosion to skin. The BCOP eye irritation study showed a score of 40.2 which is quite a high score but less than the 55 threshold for category 1 but well above the cut off for no classification of 3. With this result an in-vivo rabbit eye irritation test is required to confirm the classification, however with the classification as corrosive to skin it is not ethically acceptable to test the test substance in rabbit eyes. It is therefore concluded that the test substance in category 1 for damage to eyes.