Registration Dossier

Administrative data

Description of key information

A modern, GLP-and guideline-compliant acute oral toxicity study is available for the Registered Substance.  A modern, GLP-and guideline-compliant dermal absorption study is available for the read-across substance [Fatty acid C18 unsat, reaction products with diethylenetriamine, acetate salts].   A waiver is proposed for acute inhalation toxicity based on the availability of data for two other exposure routes and based on the physicochemical properties and use pattern of the substance.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 October to 20 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary study conducted according to relevant guidelines and GLP, with no deviations.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
The test animals were 8-10 week old healthy female Wistar Crl: WI(Han) (full barrier, SPF) rats. They were nulliparous and non-pregnant. Body weights on the day of administration ranged from 160 - 179 g. The rats were group housed in IVC cages (type III H) on Altromin saw fibre bedding, in an air-conditioned room maintained at a temperature of 22±3°C and relative humidity of 55±10%. There was a 12 hour light/dark cycle, and 10 air changes per hour. The rats were allowed free access to food (Altromin 1324 maintenance diet for rats and mice) and tap water, sulphur acidified to a pH value of approximately 2.8. Prior to test substance administration, food was withheld from the animals for 16 to 19 hours (access to water was allowed). Food was returned to the animals approximately 4 hours post-dosing. The rats were acclimatised to the laboratory conditions for at least five days.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
sterile water
Details on oral exposure:
Aqua ad injectionem (sterile water) was used as the vehicle. Homogeneity of the test material in the vehicle was maintained by vortexing the test solution for 30 minutes prior to each administration. The dosing volume was 10 mL/kg bw.
Doses:
Step 1 and 2: 2000 mg/kg bw
Step 3 and 4: 300 mg/kg bw
No. of animals per sex per dose:
3 female rats per Step
Control animals:
no
Details on study design:
The acute toxic class method was followed. The starting dose was 2000 mg/kg bw. No test material related mortality was recorded for any animal of Step 1, therefore a second step was performed at a dose of 2000 mg/kg bw. Mortality was noted at Step 2, therefore a third step was performed at 300 mg/kg bw. Based on the results obtained at Step 3, a fourth step was performed at 300 mg/kg bw. Surviving animals were observed for 14 days post-dosing. Clinical examinations were made several times on the day of dosing, at least once during the first 30 minutes and with special attention during the first 4 hours post-dose. Animals were observed for clinical signs once daily thereafter, until the end of the observation period. Body weights were recorded on Day 1 (prior to administration), and on Days 8 and 15 of the observation period. At the end of the 14 day observation period, survivors were sacrificed and subject to gross necropsy.
Statistics:
Not required.
Preliminary study:
Not applicable.
Sex:
female
Dose descriptor:
LD50
Effect level:
500 mg/kg bw
Based on:
test mat.
Remarks on result:
other: cut-off value
Mortality:
No mortalities were observed in the Step 1 rats dosed with 2000 mg/kg bw. All three rats in Step 2 (2000 mg/kg bw) were found dead between approximately 1 and 4 hours post-dosing. No mortalities were observed in the Step 3 and 4 rats (300 mg/kg bw).
Clinical signs:
Clinical signs observed in Step 1 rats within 4 hours of dosing included slightly to moderately reduced spontaneous activity, recumbency, bradykinesia, slight piloerection, hunched posture and half-eyelid closure. No signs of toxicity were noted from the day after dosing through to the end of the observation period.
Clinical signs observed in Step 2 rats within 4 hours of dosing (prior to death) included slightly to moderately reduced spontaneous activity, recumbency, bradykinesia, slight to moderate piloerection, kyphosis, catalepsis and half-eyelid closure.
Clinical signs observed in Step 3 rats within 4 hours of dosing included slightly reduced spontaneous activity, bradykinesia, slight piloerection, kyphosis and half-eyelid closure. No signs of toxicity were noted from the day after dosing through to the end of the observation period.
Clinical signs observed in Step 4 rats within 4 hours of dosing included moving the bedding, slight wasp waist, slight piloerection, kyphosis, moderately reduced spontaneous activity and half-eyelid closure. Slightly reduced spontaneous activity was observed between 4 hours post dosing and day 2. No signs of toxicity were observed from day 3 until the end of the observation period.
Body weight:
Bodyweight loss was noted in one Step 3 female (300 mg/kg bw) during week 2 of the observation period, all other surviving rats gained weight during the observation period.
Gross pathology:
Two rats in Step 2 (2000 mg/kg bw) were found to have stomachs distended with gas at necropsy. No other findings were noted.
Other findings:
No other findings reported.

No further information.

Interpretation of results:
Toxicity Category IV
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of the study, the LD50 cut-off value is considered to be 500 mg/kg bw.
Executive summary:

The acute oral toxicity of AAI_C18_PEHA_ACETATES was evaluated in female Wistar Crl: WI(Han) rats, according to the Acute Toxic Class method (OECD 423). The test material was dissolved in sterile water and administered to two groups of 3 females each at a dose of 2000 mg/kg bw, by gavage. A further two groups of 3 females each were administered a dose of 300 mg/kg bw by gavage. Animals were observed regularly within the first 4 hours after dosing for signs of toxicity and mortality. Daily observations were made for 14 days thereafter, and gross necropsy was performed on decedents or on survivors at the end of the observation period. Body weights were recorded on Day 1 (prior to administration), and on Days 8 and 15 of the observation period. There were no deaths in Step 1 animals administered 2000 mg/kg bw, however all 3 rats administered 2000 mg/kg bw in Step 2 died spontaneously on the day of dosing. No deaths were observed in the Step 3 and 4 animals administered 300 mg/kg bw. Relevant clinical findings in animals administered 2000 mg/kg bw included reduced spontaneous activity, piloerection, half eyelid-closure, bradykinesia, recumbency, catalepsis and kyphosis. All symptoms recovered by Day 2 for surviving animals. Relevant clinical findings in animals administered 300 mg/kg bw included reduced spontaneous activity, piloerection, half eyelid-closure, bradykinesia, moving the bedding, wasp waist and hunched posture. All symptoms recovered by Day 3. There were no effects on weight gain during the observation period, except for one animal which showed weight loss during the second week. No treatment-related macroscopic findings were observed.

Based on the results of the study, the LD50 cut-off value is considered to be 500 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
500 mg/kg bw
Quality of whole database:
A modern, GLP-and guideline-compliant study is available for the Registered Substance.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
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 21 September 2011 and 12 October 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study 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.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1200 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (MAFF), Testing Guidelines for Toxicology Studies, 12 NohSan No. 8147, amended 26 June 2001
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Health and Welfare, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Five male and five female Wistar (RccHan:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: Eight to twelve weeks of age.
- Weight at study initiation: At the start of the study the animals weighed at least 200g. The weight variation did not exceed ±20% of the mean weight for each sex.
- Fasting period before study: None.
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The animals were housed individually during the 24 Hour exposure period and in groups of up to four, by sex, for the remainder of the study.
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water was allowed throughout the study
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): At least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Remarks:
For the purposes of the study the test item was weighed out according to each animals individual bodyweight.
Details on dermal exposure:
TEST SITE:
On the day before treatment the back and flanks of each animal were clipped free of hair.

In the absence of data suggesting the test item was toxic, one male and one female rat were initially treated with the test item at a dose level of 2000 mg/kg.

The appropriate amount of test item was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area) using a graduated syringe. A piece of surgical gauze was placed over the treatment area and semi-occluded with a piece of self-adhesive bandage. The animals were caged individually for the 24-Hour exposure period and for the remainder of the test. Shortly after dosing the dressings were examined to ensure that they were securely in place.

After the 24-Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item.

As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg bodyweight to give a total of five males and five females. After the 24-Hour contact period the bandages were carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item. These animals were returned to group housing for the remainder of the test period.

Duration of exposure:
24 hours
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5 males and 5 females at 2000 mg/kg bodyweight.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days

- Frequency of observations and weighing:
The animals were observed for deaths or overt signs of toxicity at ½, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.

After removal of the dressings and subsequently once daily for fourteen days, the test sites were examined for evidence of primary irritation and scored according to the Draize scale (see evaluation of skin reactions).

Any other skin reactions, if present were also recorded.

Individual bodyweights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.

- Necropsy of survivors performed: yes
At the end of the study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.
Statistics:
No statistical analysis was performed.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: No deaths at the limit dose: 95% confidence limits not reported.
Mortality:
There were no deaths.


Clinical signs:
There were no signs of systemic toxicity.
Body weight:
Individual bodyweights and weekly bodyweight changes are given in Table 4.
Animals showed expected gains in bodyweight over the study period, except for three animals which showed bodyweight loss during the first week but expected gain in bodyweight during the second week.
Gross pathology:
No abnormalities were noted at necropsy.

Other findings:
Dermal Reactions:
Signs of dermal irritation noted were light brown discolouration of the epidermis, small superficial scattered scabs, hardened light brown or dark brown/black coloured scab, scab lifting to reveal glossy skin, scab lifting at edges to reveal dried blood, scab cracking, scab undulating, loss of skin elasticity and flexibility and glossy skin. Adverse reactions prevented accurate evaluation of erythema and oedema at the test sites of nine animals.

Individual clinical observations and mortality data are given in Table 1.

Individual Clinical Observations and Mortality Data

Dose Level

mg/kg bw

Animal Number and Sex

Effects Noted After Initiation of Exposure (Hours)

Effects Noted After Initiation of Exposure (Days)

½

1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

2000

1-0

Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-0

Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-1

Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-2

Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-3

Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-0

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-0

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-1

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-2

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-3

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0



0= No signs of systemic toxicity

Individual Bodyweights and Weekly Bodyweight Changes

Dose Level mg/kg

Animal Number and Sex

Bodyweight (g) at Day

Bodyweight Change (g) During Week

0

7

14

1

2

2000

1-0 Male

253

243

269

-10

26

3-0 Male

236

239

242

3

3

3-1 Male

237

241

250

4

9

3-2 Male

276

284

293

8

9

3-3 Male

277

279

283

2

4

2-0 Female

216

210

219

-6

9

4-0 Female

201

216

220

15

4

4-1 Female

220

226

231

6

5

4-2 Female

204

204

208

0

4

4-3 Female

214

210

216

-4

6

Table 5              Individual Necropsy Findings

Dose Level

mg/kg bw

Animal Number
and Sex

Time of Death

Macroscopic Observations

2000

1-0 Male

Killed Day 14

No abnormalities detected

3-0 Male

Killed Day 14

No abnormalities detected

3-1 Male

Killed Day 14

No abnormalities detected

3-2 Male

Killed Day 14

No abnormalities detected

3-3 Male

Killed Day 14

No abnormalities detected

2-0 Female

Killed Day 14

No abnormalities detected

4-0 Female

Killed Day 14

No abnormalities detected

4-1 Female

Killed Day 14

No abnormalities detected

4-2 Female

Killed Day 14

No abnormalities detected

4-3 Female

Killed Day 14

No abnormalities detected

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg bodyweight.
The test item did not meet the criteria for classification according to the Classification, Labelling and Packaging Regulation or the Globally Harmonised Classification System.
Executive summary:

. The study was performed to assess the acute dermal toxicity of the read-across substance [Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts] in the rat, according to OECD 402. Initially, two animals (one male and one female) were given a single, 24-hour, semi-occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg bw. Based on the results of the initial test, a further group of eight animals (four males and four females) was similarly treated. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy.

There were no deaths or signs of systemic toxicity. Signs of dermal irritation noted were light brown discolouration of the epidermis, scabbing, scab lifting to reveal glossy skin or dried blood, scab cracking, scab undulating, loss of skin elasticity and flexibility and glossy skin. Adverse reactions prevented accurate evaluation of erythema and oedema at the test sites of nine animals. Animals showed expected gains in bodyweight over the study period, except for three animals which showed bodyweight loss during the first week but expected gain in bodyweight during the second week. No abnormalities were noted at necropsy.  The acute dermal median lethal dose (LD50) of the test material in the rat was found to be greater than 2000 mg/kg bodyweight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw
Quality of whole database:
A modern, GLP-and guideline-compliant study is available for the read-across substance [Fatty acid C18 unsat, reaction products with diethylenetriamine, acetate salts].

Additional information

Acute oral toxicity

The acute oral toxicity of Fatty acids C18 unsat, reaction products with pentaethylenehexamine, acetate salts has been investigated in rats in a study conducted according to OECD Guideline 423 and EU Method B.1 tris using the Acute Toxic Class method (Leoni, 2012). In the study two groups of three female WISTAR Crl: WI (Han) rats were given a single dose of the test article by oral gavage at 2000 mg/kg bw in two steps. The test substance was dissolved in the vehicle (sterile water) at a concentration of 0.2 g/mL and administered at a dose volume of 10 ml/kg. Two further groups of female rats were treated with the test substance by oral gavage at a dose level of 300 mg/kg bw/day in two steps. The test substance was dissolved in sterile water at 0.03 mg/L and administered at a dose of 10 ml/kg. Three animals treated with the test substance at 2000 mg/kg bw at the second step died spontaneously on the day of treatment. All remaining animals survived until the end of the study. In animals treated at 2000 mg/kg bw/d, reduced spontaneous activity, piloerection, half eyelid closure, bradykinensia, recumbency, catalepsies and kyphosis were observed. All symptoms recovered within two days post-dose for the surviving animals. In animals treated at 300 mg/kg bw/day, reduced spontaneous activity, piloerection, half eyelid closure, bradykinesia, moving the bedding, wasp waist and kyphosis were observed. All symptoms recovered within three days of dosing for surviving animals. No treatment related macroscopic findings were observed in any animal at any step. The LD50for the acute oral toxicity of the substance was determined to be 500 mg//kg bw/d.

Acute dermal toxicity

There are no studies available on the acute dermal toxicity of Fatty acids C18 unsat, reaction products with pentaethylenehexamine, acetate salts. Based on existing datasets and structural and chemical considerations, read-across from Fatty acids C18 unsat, reaction products with pentaethylenehexamine, acetate saltsto an acute dermalstudy using Fatty acid C18 unsat, reaction products with diethylenetriamine, acetate salts is appropriate to meet the REACH Annex VII-IX data requirements. Read-across is scientifically justified and also enables the REACH requirements to be adequately addressed, while avoiding unnecessary animal testing in accordance with EU Directive 86/609/EEC. Fatty acid C18 unsat, reaction products with diethylenetriamine, acetate salts is a complex mixture of reaction products derived from the neutralisation of Fatty acid C18 unsat, reaction products with diethylenetriamine with acetic acid. The available data within the group of amidoamine/imidazoline substances indicate that for AAI substances based on shorter polyethyleneamines (EA), higher toxicity is observed compared to AAI based on longer EA. The forming of imidazoline itself does not seem to play a significant role. For cross-reading in general Fatty acid C18 unsat reaction product with diethylenetriamine therefore represents the worst case. Read-across to data on its acetate salt is therefore considered to be appropriate for evaluating the acute dermal toxicity ofFatty acids C18 unsat, reaction products with pentaethylenehexamine, acetate salts.

The acute dermal toxicity of Fatty acid C18 unsat, reaction products with diethylenetriamine, acetate salts has been investigated in a study conducted according to OECD Test Guideline 402 (Sanders, 2012). In the study two Wistar rats (one male and one female) initially were given a single 24 hour semi occluded dermal application of the test substance to intact skin at a dose of 2000 mg/kg bw. Based on the results of the initial test, a further group of eight animals (four males and four females) were administered a similar treatment. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. No deaths or clinical signs of systemic toxicity were observed in any of the treated animals. Signs of dermal irritation were observed at the application sites. No abnormalities were noted at necropsy. The acute dermal LD50of the test substance in Wistar rats was found to be greater than 2000 mg/kg bw.

Acute inhalation toxicity

A waiver is proposed for acute inhalation toxicity studies in accordance with Column 2 of Annex VIII of the REACH Regulation on the basis that acute toxicity data are available for the oral route and dermal routes of exposure.  Inhalation is not predicted to be a significant route of exposure based on the physicochemical properties of the substance. The substance is a non-volatile liquid and the use pattern indicates that significant inhalation exposure is unlikely. No additional testing is therefore warranted.


Justification for selection of acute toxicity – oral endpoint
Sole study available for this endpoint.

Justification for selection of acute toxicity – dermal endpoint
Sole study available for this endpoint.

Justification for classification or non-classification

The LD50for the acute oral toxicity of theFatty acids C18 unsat, reaction products with pentaethylenehexamine, acetate salts was determined to be 500 mg/kg bw. The substance meets the criteria for classification for acute oral toxicity as Risk Phrase (R22) 'Harmful if swallowed' according to Directive 67/548/EEC and Acute Tox Cat. 4 H302: Harmful if swallowed according to Regulation 1272/2008/EC.