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Description of key information

Modern, GLP- and guideline-compliant studies of skin and eye irritation in vivo are available for the read-across substance Fatty acid C18 unsat reaction products with diethylenetriamine, acetate salts.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
The study was performed between 31 March 2000 and 17 May 2000.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed on a read-across substance; conducted in compliance with agreed protocols, with no 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 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rabbits supplied by David Percival, Moston, Sandbach, Cheshire, Uk.
- Age at study initiation: Twelve to sixteen weeks old.
- Weight at study initiation: 2.85 to 3.04 kg.
- Housing: The animals were individually housed in suspended metal cages.
- Diet (e.g. ad libitum): Free access to food was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water was allowed throughout the study.
- Acclimation period: Minimum period of five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve limits of 17 to 23°C.
- Humidity (%): The relative humidity was set to achieve limits of 30 to 70%.
- Air changes (per hr): Approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): The lighting was controlled by a time switch to give twelve hours continuous light and twelve hours darkness,
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 ml oif test material at each test site.

Duration of treatment / exposure:
Single application for a maximum duration of 4 hours.
Observation period:
Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation.
Number of animals:
3 animals.
Details on study design:
PROCEDURE:
One rabbit was initially treated. Three suitable test sites were selected on the back of the rabbit. At each test site a quantity of 0.5 ml of the test material was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin. Each patch was secured in position with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5 cm x 4.0 cm). To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset (TUBIGRIP) and the animal was returned to its cage for the duration of the exposure period.

One patch was removed at each of three time points: 3 minutes, 1 hour and 4 hours after application. Any residual test material 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, an additional animal was treated with 0.5 ml of test material. Two patches were applied to the back of the rabbit, and were allowed to remain in contact with the skin for a period of three minutes or four hours. After consideration of the skin reactions produced in the second animal, one further animal was similarly treated with 0.5 ml of the test material.

SCORING:
Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the Draize scoring system (see below).

Any other skin reactions, if present, were also recorded.

Additional observations were made on Days 7 and 14 to assess the reversibility of skin reactions.









Irritation parameter:
erythema score
Basis:
mean
Remarks:
(animal 1)
Time point:
other: 24, 48 and 72 hours
Score:
3.7
Max. score:
4
Remarks on result:
other: 4-hour exposure period
Irritation parameter:
erythema score
Basis:
mean
Remarks:
(animal 2)
Time point:
other: 24, 48 and 72 hours
Score:
2.3
Max. score:
4
Remarks on result:
other: 4-hour exposure period
Irritation parameter:
erythema score
Basis:
mean
Remarks:
(animal 3)
Time point:
other: 24, 48 and 72 hours
Score:
4
Max. score:
4
Remarks on result:
other: 4-hour exposure period
Irritation parameter:
edema score
Basis:
mean
Remarks:
(animal 1)
Time point:
other: 24, 48 and 72 hours
Score:
4
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: 4-hour exposure period
Irritation parameter:
edema score
Basis:
mean
Remarks:
(animal 2)
Time point:
other: 24, 48 and 72 hours
Score:
2.7
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: 4-hour exposure period
Irritation parameter:
edema score
Basis:
mean
Remarks:
(animal 3)
Time point:
other: 24, 48 and 72 hours
Score:
2.3
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: 4-hour exposure period
Irritant / corrosive response data:
4-Hour Exposure Period:
The individual scores for erythema/eschar and oedema are given in Table 1 (see attached background material).
Mean values are given in Table 2 (see attached background material).

Well-defined erythema and moderate to severe oedema were noted at all treated skin sites at the 1-hour observation with moderate to severe erythema and moderate to severe oedema at the 24-hour observation and well-defined to severe erythema and slight to severe oedema at the 48 and 72-hour observations. Dermal haemorrhage was noted at one treated skin site at the 1-hour observation and at two treated skin sites at the 24, 48 and 72-hour observations. Blanching of the skin was noted at one treated skin site at the 24 and 48-hour observations. Light brown discolouration of the epidermis was noted at two treated skin sites at the 24 and 48-hour observations and at all treated skin sites at the 72-hour observation. Loss of skin elasticity and flexibility were noted at two treated skin sites at the 24, 48 and 72-hour observations.
Pale green discolouration, possibly superficial dermal necrosis, was scattered over one treated skin site at the 24, 48 and 72-hour observations. A hardened dark brown/black coloured scab or a hardened light brown coloured scab, which prevented an accurate evaluation of erythema and oedema, was noted at all treated skin sites at the 7-day observation. Glossy skin and/or reduced regrowth of fur were noted at all treated skin sites at the 14-day observation.

1-Hour Exposure Period:
The individual scores for erythema/eschar and oedema are given in Appendix I (see attached background material).

Very slight erythema and very slight oedema were noted at the treated skin site at the 24 and 48-hour observation with very slight erythema at the 72-hour observation.

The treated skin site appeared normal at the 7-day observation.

3-minute Exposure Period:
The individual scores for erythema/eschar and oedema are given in Appendix II (see attached background material).

Very slight erythema and very slight oedema were noted at two treated skin sites at the 1-hour observation. Very slight erythema was noted at all treated skin sites at the 24-hour observation, at two treated skin sites at the 48-hour observation and at one treated skin site at the 72-hour observation. Moderate desquamation was noted at two treated skin sites at the 7-day observation.

One treated skin site appeared normal at the 72-hour observation and two treated skin
sites appeared normal at the 14-day observation.


Individual scores

Observation

1h

24h

48h

72h

Mean (24-72h)

7d

14d

Erythema

2,2,2

3,3,4

4,2,4

4,2,4

3.33

?,?,?

0,0,0

Oedema

4,3,3

4,3,3

4,3,2

4,2,4

3.22

?,?,?

0,0,0

Interpretation of results:
Category 2 (irritant)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material, PR 4659 SALT, produced a primary irritation index of 6.3 and was classified as a severe irritant to rabbit skin according to the Draize classification scheme.
The test material produced positive criteria in 3/3 rabbits according to the EU labelling regulations and was classified as irritant to rabbit skin.
The results of the study show that the test material is a severe skin irritant.

Executive summary:

A study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit according to OECD 404. A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced moderate to severe erythema and moderate to severe oedema.  Other skin reactions noted were light brown discolouration of the epidermis, blanching of the skin, haemorrhage of the dermal capillaries, loss of skin elasticity and flexibility and scabbing. Glossy skin and/or reduced regrowth of fur were noted at all treated skin sites at the 14-day observation.  3 -minute and 1 -hour semi-occluded applications of the test material to the intact skin of one rabbit produced no corrosive effects. The test material produced a primary irritation index of 6.3 and was classified as a severe irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.  The test material produced positive criteria in 3/3 rabbits according to the EU labelling regulations and is therefore classified as irritant to rabbit skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Experimental start date: 27 March 2012, Experimental termination date: 17 April 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Read-across study conducted in compliance with agreed protocols, with no 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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: New Zealand White rabbits were received from Covance Research Products, Inc., Denver, PA on 14 Mar 2012
- Age at study initiation: The animals were born on 19 Nov 2011 (approximately 16 weeks old at study initiation).
- Weight at study initiation: The pretest body weight range was 2.5 - 2.7 kg.
- Housing: The animals were housed 1 per cage in suspended cages. Absorbent paper bedding was placed beneath the cages and changed at least three times per week.
- Diet (e.g. ad libitum): Fresh PMI Rabbit Chow was provided daily.
- Water (e.g. ad libitum): Water was available ad libitum.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The animal room was temperature controlled.
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle.

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
The test article (0.1 ml) was placed by syringe into the conjunctival sac that was formed by gently pulling the lower eyelid away from the eye. After installation, the lids were held together for approximately one second to insure adequate distribution of the test article. One eye of each rabbit was dosed.


Duration of treatment / exposure:
Single application.
Observation period (in vivo):
21 days
Number of animals or in vitro replicates:
3
Details on study design:
Type and Frquency of Observations:
Using a Mini-Maglite flashlight equipped with a high intensity bulb, the treated eye of each rabbit was examined for irritation of the cornea, iris and conjunctiva at 1, 24, 48 and 72 hours and again on Days 7, 14 and 21. The control eyes were observed at the same time periods. Sodium flurescein dye procedures were used 24-hour observation interval up to Day 21. The eye was examined with the aid of an ultra-violet light source.

Ocular reactions were graded according to the numerical Draize technique (see attached background material for Draize scale). Additional signs were described.

Body weights were recorded pretest and at termination.

Observations for toxicity and pharmacological effects were recorded daily. All animals were humanely sacrificed using CO2 following study termination.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
(Animal 1 - H5089/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 21 days
Remarks on result:
other: Score of 2 at 7, 14 and 21 day observations
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
(Animal 2 - H5090/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 21 days
Remarks on result:
other: Score of 2 at 7 day observation, scores of 3 at 14 and 21 day observation
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
(Animal 3 - H5091/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 21 days
Remarks on result:
other: Score of 2 at 7 day observation, scores of 3 at 14 and 21 day observation
Irritation parameter:
iris score
Basis:
mean
Remarks:
(Animal 1 - H5089/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
mean
Remarks:
(Animal 2 - H5090/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 21 days
Remarks on result:
other: Scores were unable to be determined because of chemosis at 24 and 48 hour observations. Score of 1 at 72 hour observation.
Irritation parameter:
iris score
Basis:
mean
Remarks:
(Animal 3 - H5091/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
mean
Remarks:
(Animal 1 - H5089/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
2.7
Max. score:
3
Reversibility:
fully reversible within: 21 days
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
mean
Remarks:
(Animal 2 - H5090/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
2.7
Max. score:
3
Reversibility:
not fully reversible within: 21 days
Remarks on result:
other: Scores of 3 at 7 and 14 day observations, score of 2 at 21 day observation
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
mean
Remarks:
(Animal 3 - H5091/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
2.7
Max. score:
3
Reversibility:
not fully reversible within: 21 days
Remarks on result:
other: Scores of 2 at 7, 14 and 21 day observations
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
(Animal 1 - H5089/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
3.3
Max. score:
4
Reversibility:
not fully reversible within: 21 days
Remarks on result:
other: Scores of 2 at 7 day observation, scores of 1 at 14 and 21 day observations
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
(Animal 2 - H5090/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
4
Max. score:
4
Reversibility:
not fully reversible within: 21 days
Remarks on result:
other: Scores of 3 at 7 and 14 days observations, score of 2 at 21 day observation.
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
(Animal 3 - H5091/F)
Time point:
other: Mean of scores at 24, 48 and 72 hours
Score:
4
Max. score:
4
Reversibility:
not fully reversible within: 21 days
Remarks on result:
other: Scores of 3 at 7, 14 and 21 day observations
Irritant / corrosive response data:
OCULAR FINDINGS:
Corneal opacity and conjunctival irritation, noted in three out of three eyes, persisted to Day 21.
Iritis, noted in three out of three eyes, cleared in two eyes by Day 7 and in one eye by Day 21.

See attached background material for Table 1 - Ocular findings, body weights and systemic observations.
Other effects:
SYSTEMIC OBSERVATIONS AND BODY WEIGHTS:
There were no abnormal physical signs noted during the observation period.
All animals gained weight.

See attached background material for Table 1 - Ocular findings, body weights and systemic observations.

According to OECD and OPPTS the test item is corrosive.

According to Kay & Calandra, the test item is severely irritating.

According to EU CLP classification, the test item should be classified as, Category 1 - Irreversible effects on the eye.

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to EU CLP classification, the test item should be classified as, Category 1 - Irreversible effects on the eye.
Executive summary:

The eye irritation potential of Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts was investigated in an OECD 405 study.

Three healthy New Zealand White rabbits (females), free from evidence of ocular irritation and corneal abnormalities, were dosed with Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts. The test material (0.1 mL) was placed into the conjunctival sac of one eye of each rabbit. The eyes were examined pre-test and scored by the Draize technique at 1, 24, 48 and 72 hours and again on Days 7, 14 and 21. The control eyes were observed at the same time periods. Sodium fluorescein dye procedures were used at the 24 -hour observation interval up to Day 21. Observations for toxicity and pharmacological effects were recorded at each ocular observation period and mortality observations were performed daily. The mean total scores were calculated by adding the total score for each time period and dividing by the number of animals. The degree of irritation was determined according to Kay & Calandra and classified according to the Official Journal of the European Union, on Classification, Labeling and Packaging of substances and mixtures. Body weights were recorded pretest and at termination.

Corneal opacity and conjunctival irritation, noted in three out of three eyes, persisted to Day 21. Iritis, noted in three out of three eyes, cleared in two eyes by Day 7 and in one eye by Day 21. The control eyes appeared normal at all observation periods. All animals gained weight. According to OECD and OPPTS the test item is corrosive. According to Kay & Calandra, the test item is severely irritating.  According to EU CLP classification, the test item should be classified as, Category 1 - Irreversible effects on the eye.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The skin and eye irritation potential of the Registered Substance [Fatty acids C18 unsat, reaction products with pentaethylenehexamine, acetate salts are available] are addressed by read-across to the related substance Fatty acids C18 unsat, reaction products with pentaethylenehexamine, acetate salts. Since Fatty acid C18 unsat reaction products with diethylenetriamine is considered to be the substance representing the worst case toxicity profile for amidoamine/imidazoline substances, read-across to its acetate salt is considered appropriate.

Skin irritation

The skin irritation potential of Fatty acid C18 unsat reaction products with diethylenetriamine, acetate salts has been investigated in New Zealand white rabbits in a study conducted according to OECD Test Guideline 404 (Allen, 2000). In the study, a single dose of the test substance was applied to the intact skin of three rabbits under an occluded dressing for 4 hours. The treatment producedmoderate to severe erythema and moderate to severe oedema in all rabbits. Other skin reactions noted were light brown discolouration of the epidermis, blanching of the skin, haemorrhage of the dermal capillaries, loss of skin elasticity and flexibility and scabbing. Glossy skin and/or reduced re-growth of fur were noted at all treated skin sites at the 14-day observation.Semi-occluded applications of the test material to the intact skin of one rabbitfor 3 minutes or 1 hoursproduced no corrosive effects.In the study,Fatty acid C18 unsat reaction products with diethylenetriamine, acetate saltsproduced positive criteria in 3/3 rabbits and a primary irritation index of 6.3. According to the Draize classification scheme; the substance was therefore identified as a severe irritant to rabbit skin. No corrosive effects were noted.

Eye irritation

The potential for Fatty acid C18 unsat reaction products with diethylenetriamine, acetate salts to cause eye irritation has been investigated in rabbits in a study conducted according to OECD Test Guideline 405 (Hall, 2012). In the study,three healthy New Zealand white rabbits (females), free from evidence of ocular irritation and corneal abnormalities, were treated with a dose of Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts. The test article (0.1 ml) was placed into the conjunctival sac of one eye of each rabbit. The eyes were examined pretest and scored by the Draize technique at 1, 24, 48 and 72 hours and again on days 7, 14 and 21. The control eyes were observed at the same time periods. Sodium fluorescein dye procedures were used at the 24 -hour observation interval up to day 21. Observations for toxicity and pharmacological effects were recorded at each ocular observation period and mortality observations were performed daily. The mean total scores were calculated by adding the total score for each time period and dividing by the number of animals. The degree of irritation was determined according to Kay and Calandra. Body weights were recorded pretest and at termination. Corneal opacity and conjunctival irritation, noted in three out of three eyes and persisted to day 21. Iritis, noted in three out of three eyes, cleared in two eyes by day 7 and in one eye by day 21.The control eyes appeared normal at all observation periods.All animals gained weight.Based on the findings of the study, the test substance was considered severely irritating to the eyes (Kay and Calandra). Based on read-across to the findings of this study,Fatty acids C18 unsat, reaction products with pentaethylenehexamine, acetate saltsis similarly predicted to be severely irritating to the eyes.


Justification for selection of skin irritation / corrosion endpoint:
Sole study available for this endpoint

Justification for selection of eye irritation endpoint:
Sole study available for this endpoint

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: highly irritating

Justification for classification or non-classification

Based on read-across, the substance meets the criteria for classification as Skin irritation Category 2, H315: Causes skin irritation according to Regulation (EC) No 1272/2008 and Xi (R38) 'Irritating to skin', according Directive 67/548/EEC.

Based on read-across, the substance meets the criteria for classification as Category 1 H318: Causes serious eye damage according to Regulation (EC) No 1272/2008 and Xi (R41) 'Risk of serious damage to eyes' according to Directive 67/548/EEC.

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