Reaction mass of Amines, N-tallow alkyltrimethylenedi-, mono(2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3-diaminopropane ditallate

Endpoint summary

Description of key information

Acute LD50 between 500 and 1000 mg/kgbw, based on read-across.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007-11-16 to 2008-01-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Justification for type of information:

The starting material for this reaction mass is n-tallow alkyltrimethyenediamine CAS No 61791-55-7, which was registered for REACH as N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine(CAS No 1219010-04-4), that is reacted with tall oil fatty acids (CAS No 61790-12-3), 2-ethylhexanoic acid (CAS No 149-57-5) and acetic acid (CAS No 64-18-7). The n-tallow alkyltrimethyenediamine is ca. 52.2% of the reaction mixture with the 2-ethylhexanoic acid at 10.6% compared to the tall oil fatty acids at 32.2% and the acetic acid 4.6%. The acetic acid, tall oil fatty acids and the 2-ethyhexanoic acid, have low acute oral toxicity compared to the the n-tallow alkyltrimethyenediamine. Therefore the acute oral toxicity will come essentially from the main (52.2%) component in the reaction mass, the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine. Therefore the assessment for acute oral toxicity for the reaction mass can be based on this substance. There is data on this substance but from relatively old reports, the Key study is for the close structural analogue (Z)-N-9-Octadecenylpropane-1,3-diamine CAS No 7173-62-8. This is also C16 and C18 but it is predominantly C18 and the C18 is almost all unsaturated rather than the lower level in the target molecule. The higher degree of unsaturation in the source substance increases its reactivity and therefore its acute oral toxicity, it is therefore a worst case when used as a source for read across to the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine target substance.The starting material for this reaction mass is n-tallow alkyltrimethyenediamine CAS No 61791-55-7, which was registered for REACH as N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine(CAS No 1219010-04-4), that is reacted with tall oil fatty acids (CAS No 61790-12-3), 2-ethylhexanoic acid (CAS No 149-57-5) and acetic acid (CAS No 64-18-7). The n-tallow alkyltrimethyenediamine is ca. 52.2% of the reaction mixture with the 2-ethylhexanoic acid at 10.6% compared to the tall oil fatty acids at 32.2% and the acetic acid 4.6%. The acetic acid, tall oil fatty acids and the 2-ethyhexanoic acid, have low acute oral toxicity compared to the the n-tallow alkyltrimethyenediamine. Therefore the acute oral toxicity will come essentially from the main (52.2%) component in the reaction mass, the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine. Therefore the assessment for acute oral toxicity for the reaction mass can be based on this substance. There is data on this substance but from relatively old reports, the Key study is for the close structural analogue (Z)-N-9-Octadecenylpropane-1,3-diamine CAS No 7173-62-8. This is also C16 and C18 but it is predominantly C18 and the C18 is almost all unsaturated rather than the lower level in the target molecule. The higher degree of unsaturation in the source substance increases its reactivity and therefore its acute oral toxicity, it is therefore a worst case when used as a source for read across to the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine target substance.The starting material for this reaction mass is n-tallow alkyltrimethyenediamine CAS No 61791-55-7, which was registered for REACH as N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine(CAS No 1219010-04-4), that is reacted with tall oil fatty acids (CAS No 61790-12-3), 2-ethylhexanoic acid (CAS No 149-57-5) and acetic acid (CAS No 64-18-7). The n-tallow alkyltrimethyenediamine is ca. 52.2% of the reaction mixture with the 2-ethylhexanoic acid at 10.6% compared to the tall oil fatty acids at 32.2% and the acetic acid 4.6%. The acetic acid, tall oil fatty acids and the 2-ethyhexanoic acid, have low acute oral toxicity compared to the the n-tallow alkyltrimethyenediamine. Therefore the acute oral toxicity will come essentially from the main (52.2%) component in the reaction mass, the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine. Therefore the assessment for acute oral toxicity for the reaction mass can be based on this substance. There is data on this substance but from relatively old reports, the Key study is for the close structural analogue (Z)-N-9-Octadecenylpropane-1,3-diamine CAS No 7173-62-8. This is also C16 and C18 but it is predominantly C18 and the C18 is almost all unsaturated rather than the lower level in the target molecule. The higher degree of unsaturation in the source substance increases its reactivity and therefore its acute oral toxicity, it is therefore a worst case when used as a source for read across to the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine target substance.The starting material for this reaction mass is n-tallow alkyltrimethyenediamine CAS No 61791-55-7, which was registered for REACH as N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine(CAS No 1219010-04-4), that is reacted with tall oil fatty acids (CAS No 61790-12-3), 2-ethylhexanoic acid (CAS No 149-57-5) and acetic acid (CAS No 64-18-7). The n-tallow alkyltrimethyenediamine is ca. 52.2% of the reaction mixture with the 2-ethylhexanoic acid at 10.6% compared to the tall oil fatty acids at 32.2% and the acetic acid 4.6%. The acetic acid, tall oil fatty acids and the 2-ethyhexanoic acid, have low acute oral toxicity compared to the the n-tallow alkyltrimethyenediamine. Therefore the acute oral toxicity will come essentially from the main (52.2%) component in the reaction mass, the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine. Therefore the assessment for acute oral toxicity for the reaction mass can be based on this substance. There is data on this substance but from relatively old reports, the Key study is for the close structural analogue (Z)-N-9-Octadecenylpropane-1,3-diamine CAS No 7173-62-8. This is also C16 and C18 but it is predominantly C18 and the C18 is almost all unsaturated rather than the lower level in the target molecule. The higher degree of unsaturation in the source substance increases its reactivity and therefore its acute oral toxicity, it is therefore a worst case when used as a source for read across to the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine target substance.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: HsdRccHan:WIST (Full-Barrier)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Weight at study initiation:
Step 1: 154-166 g
Step 2: 171-191 g
Step 3: 172-179 g

- Fasting period before study: overnight
- Housing: Macrolon cages on Altromin saw fiber bedding
- Diet: ad libitum, Altromin 1324
- Water: ad libitum (drinking water)
- Acclimation period: at least 5 days
-Other: marked individually by tail painting, SPF animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
Step 1: 1 g/ 5 mL and 2 g/10 mL cotton seed oil
Step 2: 0.3 g/ 10 mL cotton seed oil
Step 3: 0.3 g/ 10 mL cotton seed oil
- Amount of vehicle: 10 mL/ kg body weight
- Justification for choice of vehicle: non-toxic characteristic
- Lot/batch no.: Lot 067K0116, Sigma Chemicals

MAXIMUM DOSE VOLUME APPLIED: 1.91 mL

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: 2000 mg/ kg was selected as the starting dose

Doses:

Step 1: 2000 mg/ kg
Step 2 and 3: 300 mg/ kg body weight

No. of animals per sex per dose:

female nulliparous rats
2000 mg/ kg dose (Step 1): 3 animals
300 mg/ kg dose (Step 2 and 3): 3 animals + 3 animals

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing:
A careful clinical examination was made several times on the day of dosing.
Parts of this were at least three observations within the first four hours postdose.
Animals were observed once a day thereafter. The animals were weighed prior to
the administration and once a week thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed:
Cage side observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity
and behaviour pattern were examined. Particular attention was directed to observations of tremor,
convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period
the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross
necropsy. All gross pathological changes were recorded.

Statistics:

not applicable

Preliminary study:

no data

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

500 mg/kg bw

Remarks on result:

other: acute toxic class method; according to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4

Mortality:

Step 1 (2000 mg/kg bw): all 3 animals died within 24 h (one was euthanised due to animal welfare reasons, two were found dead in their cages)
Step 2 (300 mg/kg bw): 1 animal was found dead 10 days after treatment
Step 3 (300 mg/kg bw): no mortality observed

Clinical signs:

other: see remarks on results including tables and figures

Gross pathology:

Effect on organs (related to dose level):

Animal no. 1 of step 1 (2000 mg/ kg bw):
The animal was found dead in a lateral position. Rests of the test item were
found in the stomach. The lungs were infiltrated with blood.

Animal no. 2 of step 1 (2000 mg/ kg bw):
Rests of the test item were found in the stomach. The lungs were infiltrated
with blood.

Animal no. 3 of step 1 (2000 mg/ kg bw):
Rests of the test item were found in the stomach, which was bloated. The
small intestine was empty and of a yellow colour. This animal was
euthanised due to animal welfare reasons.

Animal no. 2 of step 2 (300 mg/ kg bw):
Stomach, small and large intestine bloated and empty.

Signs of toxicity related to dose level used, time of onset and duration:

Animal no. 1 of step 1 (2000 mg/kg bw): 30 min post-dose: salivation. 2 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure. 4 h post-dose: moderately reduced spontaneous activity, piloerection, salivation, full eyelid-closure. 5 h as well as 9 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure. 24 h post-dose the animal was found dead.

Animal no. 2 of step 1 (2000 mg/kg bw): 30 min post-dose: salivation. 2 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure. 4 h post-dose: moderately reduced spontaneous activity, piloerection, salivation, full eyelid-closure. 5 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure. 9 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure, slight articulation. 24 h post-dose the animal was found dead.

Animal No. 3 of step 1 (2000 mg/kg bw): 30 min post-dose: salivation. 2 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, salivation. 4 h post-dose: moderately reduced spontaneous activity, piloerection, salivation, full eyelid-closure. 5 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure. 9 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure, slight articulation. 24 h post-dose: moderately reduced spontaneous activity, piloerection, salivation, half eyelid-closure, severe articulation. 25 h post-dose: for animal welfare reasons this animal was euthanised.

Animal no. 1 of step 2 (300 mg/kg bw): 3 h 25 min as well as 7 h, 24 h, 30 h post-dose: piloerection. 48 h post-dose until the end of the observation period: no symptoms were observed.

Animal no. 2 of step 2 (300 mg/kg bw): 45 min as well as 2 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, half eyelid-closure. 4 h 45 min post-dose: slightly reduced spontaneous activity, piloerection. 23 h 45 min post-dose: weight loss, stertorousness. 47 h 45 min as well as 71 h 45 min, 95 h 45 min, 119 h 45 min, 143 h 45 min, 167 h 45 min, 191 h 45 min post-dose: piloerection, weight loss, stertorousness. 215 h 45 min. post-dose: moderately reduced spontaneous activity, piloerection, weight loss, stertorousness. 10 days (238 h 45 min) post-dose: the animal was found dead.

Animal no. 3 of step 2 (300 mg/kg bw): 1 h 15 min post-dose: piloerection. 2 h 45 min as well as 4 h 45 min post-dose: slightly reduced spontaneous activity, piloerection. 23 h 45 min post-dose until the end of the observation period: no symptoms were observed.

Animal no. 1, 2 and 3 of step 3 (300 mg/kg bw): 15 min as well as 1 h 45 min post-dose: slightly reduced spontaneous activity, piloerection. 3 h 45 min post-dose: moderately reduced spontaneous activity, piloerection. 21 h 45 min as well as 45 h 45 min post-dose: slightly reduced spontaneous activity, piloerection. 69 h 45 min post-dose: piloerection. 93 h 45 min post-dose until the end of the observation period: no symptoms were observed.

Absolute body weights in g

Animal No.	Sex	Day 0	Day 7	Day 14
Step 1 (2000 mg/kg bw)
1	female	154	This animal was found dead.
2	female	157	This animal was found dead.
3	female	166	This animal was found dead
Step 2 (300 mg/kg bw)
1	female	171	189	212
2	female	191	137	This animal was found dead.
3	female	173	187	200
Step 3 (300 mg/kg bw)
1	female	172	180	196
2	female	179	192	206
3	female	173	165	182

Interpretation of results:

harmful

Remarks:

Migrated information criteria given in Annex VI of the Directive 2001/59/EC Criteria used for interpretation of results: EU

Conclusions:

Considering the reported data of this toxicity test it can be stated that the test item N-Oleyl-1,3-diaminopropane showed acute oral toxic characteristics. According to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4 (LD50
cut-off: 500 mg/kg bw).

Executive summary:

The acute toxic class method for oral toxicity was performed on nulliparous female HsdRccHan:WIST rats, who received a single exposure of N-Oleyl-1,3-diaminopropane (liquid) via oral gavage.

A careful clinical examination was made several times on the day of dosing. Parts of this were at least three observations within the first four hours post-dose. Animals were observed once a day thereafter for 14 days. Cage side observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross necropsy. All gross pathological changes were recorded.

In step 1 three female rats were dosed by oral gavage with 2000 mg N-Oleyl-1,3-diaminopropane/ kg body weight in cotton seed oil. Severe toxicity indicated by salivation, apathy, piloerection and half eyelid-closure were observed in this group after the application. Two animals died within 24 h and one was euthanized 25 h post-dose for animal welfare reasons. In step 2 three female rats were dosed with 300 mg N-Oleyl-1,3-diaminopropane/ kg body weight. Two animals showed slight signs of toxicity indicated by piloerection and reduced spontaneous activity until 48 h post-dose and had recovered completely thereafter. One animal showed severe signs of toxicity indicated by reduced spontaneous activity, half eyelid-closure, piloerection, weight loss and stertorousness and was found dead after 10 days. Therefore three additional female rats were dosed with 300 mg N-Oleyl-1,3 -diaminopropane/kg body weight. Observed signs of toxicity were slightly reduced spontaneous activity and piloerection until 4 days post-dose. The animals showed no symptoms thereafter. According to the toxic class regime no further testing was required.

Following pathological changes were observed in the step 1 animals (2000 mg/kg bw): rests of the item were found in the stomach of all three rats, the lungs were infiltrated with blood in all three rats, one animal had a bloated stomach and an empty small intestine of a yellow colour. The animal of step 2 (300 mg/kg bw) that was found dead after 10 days had a bloated and empty stomach and small and large intestine.

Considering the reported data of this toxicity test it can be stated that the test item N-Oleyl-1,3-diaminopropane showed acute oral toxic characteristics. On basis of the test results and in conformity with the criteria given in Annex VI of the Directive 2001/59/EC the substance should be classified as harmful. According to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4 (LD₅₀ cut-off: 500 mg/kg bw).

The acute oral toxicity test was conducted under GLP according to the guidelines for the acute toxic class method OECD 423, EU method B1.tris and OPPTS 870.110 without deviation.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1975

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Substance identification: "EC 63". Otherwise standard acute oral limit test, but no indication of QA or GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Principles of method if other than guideline:

The used dose volume of 40 ml/kg is very high and also the dose given is far above the limit dose (16 g/kg). No necropsy was performed on the surviving animals.

GLP compliance:

no

Remarks:

the study was performed pre-GLP

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: CFY

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data besides that weight of the animals was between 95-114g

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

EC 63 was prepared as a 40% suspension in corn oil and administered by oral intubation at a dosage volume of 40 ml/kg bodyweight. Animals were staved overnight before treatment.

Doses:

Range finding study: 1, 4, 16 g kg/bw
main study: 16 g/kg bw

No. of animals per sex per dose:

Range finding study: 2
main study: 5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: body weight are reported at day of dosing, after 1 week and after 2 weeks
- Necropsy of survivors performed: yes
- Other examinations performed: none

Statistics:

None

Preliminary study:

In the preliminary study one animal died in the 4 g/kg bw group within 42 hours of treatment. Autopsy reveated pallor of the liver and injection of blood vessels at the peritoneum. However, as no other rats showed severe toxic effects at this or higher levels, it was considered that this mortality did not indicate general toxicity of this treatment level.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 16 000 mg/kg bw

Mortality:

No deaths occured

Clinical signs:

other: Signs of reaction to treatment observed shorlty after dosing, consisted of lethargy, piloerection and a slight increase in salivation. recovery as judged by appearence and behavior was complete within 8 days.

Gross pathology:

Not performed.

Interpretation of results:

practically nontoxic

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In the experiment none of the exposed animals died and the LD50 of EC 63 in rats found found to be > 16 g/kg bw

Executive summary:

In a limit test 5 male and 5 female rats were exposed to 16 g/kg bw test substance in a single oral dose by gavage in corn oil. None of the animals died. In the experiment non of the exposed animals died and the LD50 of EC 63 in rats found found to be > 16 g/kg bw.

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Substance indentification limited to CESIO nr. and generic category name.

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

Version / remarks:

Commision Directive 84/449/EEC

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bantin & Kingman Ltd., Grimston, A1 dborough, Hull, U. K.
- Age at study initiation: five to eight weeks old
- Weight at study initiation: the males weighed 120 - 172g, and the females 120 - 1469
- Fasting period before study: overnight fast immediately before dosing and for approximately two to 3.5 hours after dosing.
- Housing:The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with sawdust bedding.
- Diet (e.g. ad libitum): ad libitum, except during fasting period
- Water (e.g. ad libitum):ad libitum, except during fasting period
- Acclimation period: a minimum acclimatisation period of at least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness.


IN-LIFE DATES: Not specified

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: Not specified
- Amount of vehicle (if gavage): Not specified
- Justification for choice of vehicle: Not specified
- Lot/batch no. (if required): Not specified
- Purity: Not specified

MAXIMUM DOSE VOLUME APPLIED: Not specified

DOSAGE PREPARATION (if unusual): Not specified, concentration, homogeneity and stability of the test material formulations were not determined.

- Rationale for the selection of the starting dose: range finding study

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed for deaths and overt signs of toxicity 1 and 4
hours a f t e r dosing and subsequently once daily for 14 days. Individual bodyweights were recorded on the day o f treatment (Day 0),
on Days 7 and 14 or a t death.
- Necropsy of survivors performed: A t the end of the study the animals were killed by cervical dislocation
and the abdominal and thoracic cavities opened for examination of all major organs. The macroscopic- appearance of any abnormal tissues
was recorded. No tissues were retained.
- Other examinations performed: No

Statistics:

Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test material was made. Clinical observations,
bodyweight and necropsy records were examined for any adverse but non-lethal effects resulting from treatment.

Preliminary study:

In a preliminary study one male and one female animal were administered a single dose of 500, 1000, 3000 or 5000. In the 5000 mg/kg bw group the male animal died on day 4. Based on these results 5000 mg/kg was selected for the main study.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Mortality:

One male was found dead five days after treatment and one female was found dead two days after treatment. No other deaths occurred.

Clinical signs:

other: Common signs of hunched posture and pilo-erection were noted one to ten days after treatment. Red/brown staining around the snout was also commonly noted three to five days after treatment. An isolated incident of pallor of the extremities was noted in on

Gross pathology:

Abnormalities noted at necropsy of animals that died during the study were abnormally red lungs, dark liver or patchy pallor of the liver and
haemorrhage of the gastric mucosa.
No abnormalities were noted at necropsy of animals that were killed at the end o f the study.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

LD50 > 5000 mg/kg bw

Executive summary:

The acute oral median lethal dose (LD50) of the test material in the Sprague-Dawley strain rat was found to be greater than 5000 mg/kg bodyweight. No symbol and risk phrase are required according to EEC labelling regulations.

Reference 4

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Substance identification limited to: "EC 126". Group size small (2 m/2f). Carried out before QA/GLP.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Too few animals (2 sex/dose), dose volume very high 40 ml/kg.

GLP compliance:

no

Remarks:

study was performed pre-GLP

Test type:

standard acute method

Species:

rat

Strain:

other: CFY

Sex:

male/female

Details on test animals or test system and environmental conditions:

Weight of the animals was 92-112g, no further information.

Route of administration:

oral: gavage

Vehicle:

other: aqueous methyIcellulose (1 %)

Details on oral exposure:

EC 126 was prepared as a 40% w/v suspension in aqueous methyIcellulose (1 %) and administered by oral intubation at a range of dosage volumes of 2.5 to 40ml/kg bodyweight. Rats dosed with the vehicle alone (40ml/kg) served as controls.

Doses:

1.0, 4.0 and 16.0 g/kg bw

No. of animals per sex per dose:

2

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: body weight are reported at day of dosing, after 1 week and after 2 weeks
- Necropsy of survivors performed: yes
- Other examinations performed: none

Statistics:

None

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 16 000 mg/kg bw

Mortality:

No mortalities observed

Clinical signs:

other: Signs of reaction to treatment observed shortly after dosing in both treated and control animals included piloerection and abnormal body carriage (hunched posture), these signs were accompanied by lethargy in three rats at 16g/kg and by increased salivati

Gross pathology:

Terminal autopsy findings were normal.

Interpretation of results:

practically nontoxic

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The number of animals is this study is too low to draw any firm conclusions, but based on these results the LD50 of EC 126 in rats found found to be > 16 g/kg bw.

Executive summary:

A single exposure of 2 male and 2 female rats to a single dose of 0, 1.0, 4.0 or 16.0 g/kg bw did not result in any lethalities. The number of animals is this study is too low to draw any firm conclusions but based on these results the LD50 of EC 126 in rats found found to be > 16 g/kg bw

Reference 5

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Substance identification limited to "E28". Valid provided that evidence of identity can be provided.

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, FRG
- Age at study initiation: 9 weeks
- Weight at study initiation: no data
- Fasting period before study: overnight
- Housing: individually housed in polycarbonate cages containing purified sawdust (Woody Clean supplied by Broekman Institute, Someren,
The Netherlands) as bedding material.
- Diet (e.g. ad libitum): standard pelleted laboratory animal diet (RMH-B, Hope Farms, Woerden. The Netherlands), ad libitum
- Water (e.g. ad libitum): tap-water, ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 55-80
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES:
Test substance received: October 7, 1987
Initiation date : October 14. 1987 (pilot study)
Completion date : November 11, 1987 (main study)

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 5ml/kg bw

Doses:

range finding study 1: 56, 130, 240 and 420 mg/kg bw
range finding study 2: 750, 1250 and 1800 mg/kg bw
main study: 600, 825 and 1300 mg/kg bw

No. of animals per sex per dose:

range finding study: 1 male and 1 female
main study: 5 male and 5 female

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Cage-side observations were performed on the day of dosing (approximately once every two hours) and once daily thereafter for 14 days. Any signs of toxicity were recorded along with the time of onset and duration. Individual body weights were measured weekly and at death (from day 1 of dosing), At the end of the study (day 14) all animals were killed by CO2-asphyxiation and subjected to necropsy.
- Necropsy of survivors performed: yes
- Other examinations performed: none

Statistics:

LD50 was calculated according to the "maximum likelihood method" (Finney)

Preliminary study:

In order to establish an appropriate dose range initially, four groups of animals, each comprising 1 male and 1 female, were dosed with an oral dose of the test substance at 56, 130, 240 and 420 mg/kg body weight, respectively. In order to obtain accurate dosing, the test substance was diluted with reverse osmosis water and administered in a dose volume of 5 ml/kg body weight. Since no mortalities occurred and as of day 1 no signs of toxicity were observed, three additional groups were dosed with undiluted test substance at 750, 1250 and 1800 mg/kg body weight, respectively. All animals of the 1250 and 1800 mg/kg group died on the day of dosing, Signs of toxicity, observed on the day of dosing in the 420 and 750 mg/kg group were lethargy, piloerection and pale skin. In addition, animals of the 750 mg/kg group showed cyanosis and convulsions

Sex:

male/female

Dose descriptor:

LD50

Effect level:

873 mg/kg bw

95% CL:

736 - 1 088

Sex:

male

Dose descriptor:

LD50

Effect level:

942 mg/kg bw

95% CL:

833 - 1 235

Sex:

female

Dose descriptor:

LD50

Effect level:

789

Remarks on result:

other: There was insufficient data to calculate a 95% confidence interval for females alone.

Mortality:

The incidence of mortalities for the sexes combined from low to high dose group was 1, 4 and 8. All deaths occurred within 2% hours of
dosing.

Clinical signs:

other: Signs of toxicity were lethargy, ataxia, piloerection and dark red/purple eyes. All signs were only detectabel shortly after dosing.

Gross pathology:

Macroscopic abnormalities of animals at necropsy were red glandular stomach, haemorrhages in the glandular stomach, dark red liver and
light brown lungs. Observed in one animals in the low dose group, four animals in the mid dose group and 8 animals of the high dose group.

Interpretation of results:

harmful

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

LD50 (95% confidence interval )
male and female: 873 mg/kg bw (736-1088)
males: 942 mg/kg bw (833-1235) mglkg.
females: 789 mg/kg bw (insufficient data to calculate a 95% confidence interval)

Executive summary:

The acute oral toxicity of the test substance E28 was evaluated in the rat according to OECD401 and under GLP. Three groups of Wistar rats, each comprising 5 males and 5 females, received a single oral dose of E28 at 600, 825 and 1100 mglkg body weight, respectively. The incidence of mortalities for the sexes combined from low to high dose group was 1, 4 and 8. All deaths occurred within 2.5 hours of dosing. Signs of toxicity were lethargy, ataxia, piloerection and dark redlpurple eyes. All surviving animals revealed body weight gain. Macroscopic abnormalities of animals at necropsy were red glandular stomach, haemorrhages in the glandular stomach, dark red liver and light brown lungs. The LD50 value for the sexes combined amounted to 873 mglkg body weight.

Reference 6

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Limited reporting, only a summary is available (2 pages). Study is not GLP. No information on the test substance identity. 30ml was given in a single dose to one animal which is a very high dose volume.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Principles of method if other than guideline:

30ml was given in a single dose to one animal which is a very high dose volume.

GLP compliance:

no

Remarks:

study was performed pre-GLP

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Doses:

6.0g/kg bw

No. of animals per sex per dose:

10 male and 10 female

Control animals:

no

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 6 000 mg/kg bw

Within a few hours after treatment the rats showed sedation and signs of ataxia. Later on coma was frequently observed. After 24 hours these phenomena had disappeared but the rats showed rough roats, encrustations around eyes and nortrils and signs of unthriftiness. One male and one female rat died on the fourth post-treatment day. Thereafter the survivors gradually recovered, and looked quite healthy again at the end of the observation period. Macroscopic examination of the survivors at autopsy did not reveal any treatment-related gross alterations.

Interpretation of results:

practically nontoxic

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

From these results it appears that the LD50 of DC 63 exceeds 6.0 g per kg body weight, Therefore, the test material can be classified as practically non-toxic.

Reference 7

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1972

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Summary based on abstract. Full study report not available.

Qualifier:

no guideline followed

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Species:

other: Duck

Strain:

other: Mallard

Sex:

male

Route of administration:

oral: capsule

Vehicle:

other: gelatin capsule

Doses:

Four groups. No further data.

No. of animals per sex per dose:

3 males per dose

Control animals:

not specified

symptoms observed included polydipsia, regurgitation, slowness, ataxia, goose-stepping ataxia, stumbling, falling, sitting, hyporeactivity, mydriasis, using wings to aid pedestrian locomotion, tremors, mild tetanic seizures of the legs, mild ataraxia, asthenia and emaciation. Treatment levels as low as 1000 mg/kg produce mortalities. Symptoms of intoxication (other than polydipsia and regurgitation) appeared the day after treatment and mortalities occurred usually between 1 and 11 days after treatment. Symptoms persisted for up to 16 days. Mortalities had lost a mean of 247g of body weight by death, and survivors had gained or lost small mounts of body weight by the end of the observation period.

No gross pathological changes attributable to the action of Duoneen T-E-9 were observed in tissues of the sacrificed

survivors.

Conclusions:

Not assignable. The data is too limited to draw any conclusions.

Reference 8

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1972

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Summary based on abstract. Full study report not available.

Qualifier:

no guideline followed

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Species:

other: Ring-necked pheasant

Sex:

male

Route of administration:

oral: capsule

Vehicle:

other: gelatin capsule

Doses:

Four groups. No further data.

No. of animals per sex per dose:

3 males per dose

Control animals:

not specified

Symptoms observed included fluffed feathers, hyporeactivity, ataraxua, ptosism withdrawal, ataxia, goose-stepping ataxia, slowness, imbabalnce, tremors, spasms, loss of righting reflex, emaciation, myasthenia, asthenia and immobility. One bird showed regurgitation on the day following treatment. Symptoms appeared on the day following treatment and deaths occured usually between 1 -9 days after treatment. Symptoms persisted for up to 10 days. Mortalities had lost a mean of 307g of bodyweight by death; survivors had gained normal amounts of bodyweight by the end of the observation period. Several instances of small spleens were observed in tissues of mortalities. No gross pathological chnages attributable to the action of Duomeen T-E-9 were observed in the tissues of the sacrificed survivors.

Conclusions:

Not assignable. The data is too limited to draw any conclusions.

Reference 9

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Tested Duomeen T, however no CoA available. No GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

no

Test type:

standard acute method

Species:

rat

Strain:

other: HC/CFY (Remote Sprague-Dawley)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hacking and Churchill Limited, Huntingdon, Cambridgeshire, England.
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 94 to 130 g
- Fasting period before study: Access to food only was prevented overnight prior to and approximately 4 hours after dosing .
- Housing: The r a t s were randomly allocated to cages within treatment groups. They were housed in groups by sexmetal cages with wire mesh floors
- Diet (e.g. ad libitum): Laboratory Diet No. 1, Spratt's Rodent Breeding Diet (LAD 1), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: a minimum period of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 47
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: No data

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
dose concentration
(mg/kg) (w/v %)
320 3.2
500 5.0
800 8.0
1260 12.6
- Amount of vehicle (if gavage): no data
- Justification for choice of vehicle: no data
- Lot/batch no. (if required): no data
- Purity: no data

MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg

DOSAGE PREPARATION (if unusual): The test substance was prepared on the day of dosing. The stability and absorption of the test substance were not determined.

Doses:

Preliminary study: 100, 500, 1000 and 5000 mg/kg
Main study: 320, 500, 800, 1260 mg/kg

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals on the preliminary study were observed for 5 or 14 days after dosing. The animals on the main study were observed for 14 days after dosing. Animals were observed soon after dosing; then at frequent intervals for the remainder of Day 1. On
subsequent days the animals were observed at least twice. Clinical signs were recorded at each observation.
- Other examinations performed:
The following were recorded on the main study: Approximate time of death of individual rats. The nature, severity, approximate time of onset and duration of each toxic sign. Individual bodyweights of rats on Days 1 (day of dosing), 8 and 15 and at death.

Surviving animals on the main study were killed on Day 15 by cervical dislocation. All animals that died during the study and those killed on Day 15 were subjected to a macroscopic post mortem examination which consisted of opening the abdominal and thoracic cavities. The macroscopic appearance of abnormal organs was recorded.

Statistics:

The acute median lethal oral dose (LD50) to male and female rats was calculated using the method of:
Finney (1971) Probit Analysis (3rd edition) Cambridge University Press.
Separate LD50 values for males and females were estimated by undertaking probit analysis on the mortality data by fitting two
parallel lines on the data (males only and females only) using the technique described by Finney (1978, Statistical Method in Biological
Assay, 3rd Edition, Charles Griffin, London). A chi-squared test was carried out to check that the data did not contain any evidence for
non-parallelism.

Preliminary study:

The results of the preliminary study indicated that the acute median lethal oral dose (LD5O) was in the region of 500 mg/kg bodyweight.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

945 mg/kg bw

95% CL:

741 - 1 409

Sex:

male

Dose descriptor:

LD50

Effect level:

1 186 mg/kg bw

95% CL:

818 - 2 159

Sex:

female

Dose descriptor:

LD50

Effect level:

783 mg/kg bw

95% CL:

549 - 1 140

Mortality:

Mortalities occurred in male rats dosed at 1260 mg/kg and female rats dosed at 500 rng/kg and above. These deaths were observed from within
two and five days of dosing.

Clinical signs:

other: Signs of reaction to treatment observed shortly after dosing in all treated rats included pilo-erection, abnormal body carriage (hunched posture), abnormal gait (waddling), increased salivation and pallor of the extremities. These signs were accompanied b

Gross pathology:

No abnormal findings were seen at terminal autopsy.

preliminary study

	mortality ratio (no deaths/no dosed)
dose (g/kg)	male	female	combined	time of death after dosing (hours)
100	0/2	0/2	0/4
500	1/4	3/4	4/ 8	<27 -<75
1000	0/2	1/2	1/4	<77
5000	2/2	2/2	4/4	<2 -<46

main study

sex	dose (mg/kg)	No of deaths per 5	day
			1							2		3		4		5		6 -15
			hours after dosing
			0.5	1	2	3	4	5	6	a	b	a	b	a	b	a	b	a	b
male	3205008001260	0004								0001	0002			0001
female	3205008001260	0143								0112	0020	0010				0001

a First observation

b Second observation

Interpretation of results:

harmful

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on these results the substance is classified as harmful.

Reference 10

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: There is no information about of the substance compostion and stability.

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

no

Remarks:

but inhouse QA was in place and the study and report were audited

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: HC/CFY (remote Sprague-Dawley)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hacking and Churchill Limited, Huntingdon, Cambridgeshire,
England.
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 108-140 g
- Fasting period before study: overnight before dosing and 4 hours after dosing
- Housing: They were housed in groups by sex in metal cages with wire mesh floors
- Diet (e.g. ad libitum): A standard laboratory rodent diet (Scientific Feeds LAD 1 obtained from Special Diet Services Ltd., Witham, Essex, England),ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 40
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: The study was undertaken between 3 and 17 January 1985.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

VEHICLE
substance was dosed as received

MAXIMUM DOSE VOLUME APPLIED:
5.8 ml/kg

Doses:

range finding: 1.0 and 5.0 g/kg bw
main study: 5.0 g/kg bw

No. of animals per sex per dose:

range finding: 2 male and 2 female rats
main study: 5 male and 5 female rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 5 days in the range finding study, 14 days in the main study
- Frequency of observations and weighing: Animals were observed soon after dosing; then at frequent intervals for the remainder of Day 1. On
subsequent days the animals were observed at least twice per day (The nature, severity , approximate time of onset and duration of each toxic sign). Clinical signs were recorded at each observation. Individual bodyweights of rats on days 1 (day of dosing ) , 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: none

Statistics:

None

Preliminary study:

No deaths occured

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Mortality:

None

Clinical signs:

other: Signs of reaction to treatment observed shortly after dosing were pilo-erection and abnormal body carriage (hunched posture ). Recovery as judged by external appearance and behaviour was apparently complete by Day 4 in males and Day 5 in females.

Gross pathology:

No effects observed

Interpretation of results:

practically nontoxic

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

LD50 > 5000 mg/kg bw

Executive summary:

According to and OECD401 acute oral toxicity study rats were given a single limit dose of 5000 mg/kg bw. No deaths occured and the only signs observed were observed shortly after dosing were pilo-erection and hunched posture. Recovery as judged by external appearance and behaviour was apparently complete by Day 4 in males and Day 5 in female. Therefore the LD50 is > 5000 mg/kg bw.

Reference 11

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007-11-21 to 2008-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: HsdRccHan: WIST (Full-Barrier)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Weight at study initiation:
Step 1: 168 g (1 animal)
Step 2: 170-186 g
Step 3: 141-177 g
Step 4: 135-158 g
Step 5: 128-131 g

- Fasting period before study: overnight and 3-4 hours after dosing
- Housing: macrolon cages on Altromin saw fibre bedding
- Diet: ad libitum (Altromin 1324 (TPF)
- Water: ad libitum (drinking water)
- Acclimation period: at least 5 days
- Other: marked individually by tail painting, SPF animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
Step 1: 1.0 g/ 5 mL
Step 2: 0.3 g/ 10 mL
Step 3: 0.3 g/ 10 mL
Step 4: 0.1 g/ 20 mL
Step 5: 0.1 g/ 20 mL
- Amount of vehicle: 10 mL/ kg body weight
- Justification for choice of vehicle: non-toxic and lipophil
- Lot/batch no. : Lot 067K0116, Sigma Chemicals

MAXIMUM DOSE VOLUME APPLIED: 1.86 mL

DOSAGE PREPARATION : freshly mixed prior to administration

CLASS METHOD
- Rationale for the selection of the starting dose:
preliminary test with one animal performed (2000 mg/kg), due to immeadiate preacute lethality
the starting dose was choosen to be 300 mg/kg bw

Doses:

Step 1: 2000 mg/kg bw
Step 2: 300 mg/kg bw
Step 3: 300 mg/kg bw
Step 4: 50 mg/kg bw
Step 5: 50 mg/kg bw

No. of animals per sex per dose:

Step 1 (2000 mg/kg bw): 1 female rat
Step 2 (300 mg/kg bw): 3 female rats
Step 3 (300 mg/kg bw): 3 female rats
Step 4 (50 mg/kg bw): 3 female rats
Step 5 (50 mg/kg bw): 3 female rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: A careful clinical examination was made several times on the day of dosing.
Parts of this were at least three observations within the first four hours post-dose.
Animals were observed once a day thereafter.
The animals were weighed prior to the administration and once a week thereafter.

- Necropsy of survivors performed: yes

- Other examinations performed: Cage side observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity
and behaviour pattern were examined. Particular attention was directed to observations of tremor,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

not applicable

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

300 mg/kg bw

Remarks on result:

other: acute toxic class method; according to GHS (Globally Harmonized Classification System) the test item N-coco-1,3-diaminopropane was classified into Category 3

Mortality:

Step 1 (2000 mg/kg bw, 1 female rat): within 1 h 30 min post-dose the animal was found dead
Step 2 (300 mg/kg bw): no deaths observed within the observation period
Step 3 (300 mg/kg bw): Animal 2 was found dead five days and animal 3 three days post-dose
Step 4 and 5 ( 50 mg/kg bw): no deaths observed within the observation period

Clinical signs:

other: see remarks on results including tables and figures

Gross pathology:

Animal no. 1 of step 1 (2000 mg/ kg bw):
The stomach showed haemorrhage with a foamy content. The small intestine
also showed haemorrhage with a bloody content. The large intestine and appendix were bloody.
Animal no.2 of step 3 (300 mg/ kg bw):
The animal was found dead in a lateral position and exhibited piloerection.The anus was smeared with
faeces.
Animal no. 3 of step 3 (300 mg/ kg bw):
The stomach was bloody. The small intestine was slightly bloody.

Signs of toxicity related to dose level used, time of onset and duration:

Animal no. 1 of step 1 (2000 mg/kg bw): 30 min post-dose: moderately reduced spontaneous activity, salivation, face down position.1 h 30 min post-dose: this animal was found dead.

Animal no. 1 of step 2 (300 mg/kg bw): 25 min post-dose: slightly reduced spontaneous activity, apathy, piloerection.1 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, half eyelid-closure. 5 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, sunken flank. 22 h 45 min as well as 28 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, sunken flank, diarrhoea. 46 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, diarrhoea, bloody snout, bloody mouth. 50 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, diarrhoea, weight loss. 78 h 15 min post-dose: piloerection, diarrhoea, weight loss. 98 h 10 min post-dose: slightly reduced spontaneous activity, piloerection, diarrhoea, heavy breathing (rattling). 119 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, apathy, weight loss. 143 h 45 min post-dose: apathy, piloerection.166 h 45 min post-dose: weight gain.

Animal No. 2 and 3 of step 2 (300 mg/kg bw): 30 min post-dose: slightly reduced spontaneous activity, apathy, salivation. 2 h 15 min post-dose: slightly reduced spontaneous activity (animal no. 3), moderately reduced spontaneous activity (animal no. 2), piloerection, half eyelid-closure. 4 h 15 min post-dose: moderately reduced spontaneous activity, piloerection. 23 h 15 min post-dose: slightly reduced spontaneous activity, piloerection. 28 h 15 min post-dose: moderately reduced spontaneous activity, piloerection, half eyelid-closure. 2 days post-dose until the end of the observation period: no further symptoms were observed.

Animal no. 1 of step 3 (300 mg/kg bw): 15 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 2 h as well as 4 h 30 min, 22 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure. 47 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure, diarrhoea, bloody snout, anal discharge. 71 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, diarrhoea, weight loss. 97 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, diarrhoea, weight loss. 121 h post-dose: slightly reduced spontaneous activity, piloerection, diarrhoea. 144 h post-dose: slightly reduced spontaneous activity, piloerection. 7 days post-dose until the end of the observation period: no further symptoms were observed.

Animal no. 2 of step 3 (300 mg/kg bw): 15 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 2 h as well as 4 h, 22 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure. 47 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure, diarrhoea, anal discharge. 71 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, diarrhoea, weight loss. 97 h post-dose: severely reduced spontaneous activity, piloerection, diarrhoea, weight loss. 5 days post-dose the animal was found dead.

Animal no.3 of step 3 (300 mg/kg bw): 15 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 2 h as well as 4 h, 22 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure. 47 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure, diarrhoea, anal discharge, bloody snout.3 days post-dose the animal was found dead.

Animal no. 1, 2 and 3 of step 4 and 5 (50 mg/kg bw): No compound related mortality was recorded for any animal.

Absolute body weights in g :

Animal No.	Sex	Day 0	Day 7	Day 14
Step 1 (2000 mg/kg bw)
1	female	168	This animal was found dead.
Step 2 (300 mg/kg bw)
1	female	170	150	190
2	female	180	189	205
3	female	186	196	213
Step 3 (300 mg/kg bw)
1	female	177	180	201
2	female	141	92-this animal was found dead
3	female	149	This animal was found dead.
Step 4 (50 mg/kg bw)
1	female	135	138	142
2	female	158	191	206
3	female	158	183	197
Step 5 (50 mg/kg bw)
1	female	128	162	177
2	female	131	161	176
3	female	130	163	174

Interpretation of results:

toxic

Remarks:

Migrated information criteria given in Annex VI of the Directive 2001/59/EC Criteria used for interpretation of results: EU

Conclusions:

Considering the reported data of this toxicity test it can be stated that the test item N-Coco-1,3-diaminopropane showed acute oral toxic
characteristics.
According to GHS (Globally Harmonized Classification System) the test item N-Coco-1,3-diaminopropane was classified into Category 3
(LD50 cutoff:300 mg/kg bw).

Executive summary:

The acute toxic class method for oral toxicity was performed on nulliparous female HsdRccHan:WIST rats, who received a single exposure of N-Coco-1,3-diaminopropane via oral gavage.

A careful clinical examination was made several times on the day of dosing. Parts of this were at least three observations within the first four hours post-dose. Animals were observed once a day thereafter for 14 days. Cage side observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross necropsy. All gross pathological changes were recorded.

In step 1 single female rat was dosed by oral gavage with 2000 mg N-coco-1,3-diaminopropane/ kg body weight in cotton seed oil. Severe, immediate toxicity indicated by salivation, face down position and moderately reduced spontaneous activity was observed in this animal, followed by death at 1 h and 30 min post-dose. In step 2 three female rats were dosed with 300 mg N-coco-1,3-diaminopropane/ kg body weight. Animal 1 showed severe signs of toxicity indicated by immediate piloerection followed by half eyelid closure, sunken flank, diarrhoea, bloody snout and mouth, weight loss and heavy breathing . 7 days post-dosing the animal gained weight again and recovered. Animal 2 and 3 showed reduction in spontaneous activity, apathy, salvation, piloerection and half eyelid-closure shortly after dosing. 2 days post-dose until the end of the observation period no further symptoms were evident. In step 3 three female rats were dosed with 300 mg N-coco-1,3 -diaminopropane/ kg body weight. Observed signs of toxicity of animal 1 were reduced spontaneous activity, piloerection, apathy, half eyelid-closure, diarrhoea, bloody snout, anal discharge and weight loss until 7 days post-dose. The animal showed no symptoms thereafter. Animal 2 of step 3 displayed increasing signs of toxicity indicated by reduced spontaneous activity, piloerection, apathy, half eyelid-closure, diarrhoea, anal discharge as well as weight loss and was found dead on day 5 post-dose. Animal 3 of step 3 displayed increasing signs of toxicity indicated by reduced spontaneous activity, piloerection, apathy, half eyelid-closure, diarrhoea, bloody snout, anal discharge and weight loss and was found dead on day 3 post-dose. Animal no. 1, 2 and 3 of step 4 and 5 received 50 mg N-coco-1,3-diaminopropane/ kg body weight by oral gavage. No compound related mortality was recorded for any of the animals.

According to the toxic class regime no further testing was required.

Following pathological changes were observed in the step 1 animal (2000 mg/kg bw): The stomach showed haemorrhage with a foamy content. The small intestine also showed haemorrhage with a bloody content. The large intestine and appendix were bloody. Animal 2 of step 3 (300 mg/kg bw) that was found dead after 5 days in a lateral position exhibited piloerection. Its anus was smeared with faeces. Animal no. 3 of step 3 (300 mg/ kg bw) had a bloody stomach. Its small intestine was slightly bloody.

Considering the reported data of this toxicity test it can be stated that the test item N-coco-1,3-diaminopropane showed acute oral toxic characteristics. On basis of the test results and in conformity with the criteria given in Annex VI of the Directive 2001/59/EC the substance should be classified as toxic. According to GHS (Globally Harmonized Classification System) the test item N-Coco-1,3-diaminopropane was classified into Category 3 (LD₅₀ cut-off: 300 mg/kg bw).

The acute oral toxicity test was conducted under GLP according to the guidelines for the acute toxic class method OECD 423, EU method B1.tris and OPPTS 870.110 without deviation.

Reference 12

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007-11-27 to 2007-12-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: HsdRccHan: WIST (SPF)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Weight at study initiation:
Step 1: 172-180 g
Step 2: 176-177 g

- Fasting period before study: overnight and 3-4 hours after dosing
- Housing: macrolon cages on Altromin saw fibre bedding
- Diet: ad libitum (Altromin 1324 (TPF)
- Water: ad libitum (drinking water)
- Acclimation period: at least 5 days
- Other: marked individually by tail painting, SPF-animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
Step 1: 1.0 g/ 5 mL
2.0 g/ 10 mL
Step 2: 2.0 g/ 10 mL

- Amount of vehicle: 10 mL/ kg body weight
- Justification for choice of vehicle: non-toxic and lipophil
- Lot/batch no. : Lot 067K0116, Sigma Chemicals

MAXIMUM DOSE VOLUME APPLIED: 1.80 mL

DOSAGE PREPARATION : freshly mixed to homogeneity prior to administration

CLASS METHOD
- Rationale for the selection of the starting dose:
the starting dose was choosen to be 2000 mg/kg bw

Doses:

Step 1: 2000 mg/kg bw
Step 2: 2000 mg/kg bw

No. of animals per sex per dose:

Step 1 (2000 mg/kg bw): 3 female rats
Step 2 (2000 mg/kg bw): 3 female rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: A careful clinical examination was made several times on the day of dosing.
Parts of this were at least three observations within the first four hours post-dose.
Animals were observed once a day thereafter.
The animals were weighed prior to the administration and once a week thereafter.

- Necropsy of survivors performed: yes

- Other examinations performed: Cage side observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity
and behaviour pattern were examined. Particular attention was directed to observations of tremor,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

not applicable

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

2 500 mg/kg bw

Remarks on result:

other: acute toxic class method; according to GHS (Globally Harmonized Classification System) the test item N-(Hydrogenated tallow)-1,3-diaminopropane was classified into Category 5

Mortality:

Step 1 (2000 mg/kg bw): 28 h post-dose one animal was found dead

Clinical signs:

other: see remarks on results including tables and figures

Gross pathology:

Animal no. 3 of step 1 (2000 mg/ kg bw):
The dead animal was found in a lateral position. Stomach: solid content.
Small and large intestine: partly filled with solid substance.
Animal no.2 of step 2 (2000 mg/ kg bw):
The spleen was enlarged, with a weight of 0.872 g.

Signs of toxicity related to dose level used, time of onset and duration:

Animal no. 1 of step 1 (2000 mg/kg bw): 20 min as well as 125 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 21 h 05 min as well as 27 h 5 min, 45 h 05 min post-dose: slightly reduced spontaneous activity, piloerection. 51 h 5 min post-dose: slightly reduced spontaneous activity. 69 h 05 min until the end of the observation period: no further symptoms were observed.

Animal no. 2 of step 1 (2000 mg/kg bw): 30 min as well as 3 h post-dose: slightly reduced spontaneous activity, apathy, piloerection. 24 h as well as 45 h post-dose: slightly reduced spontaneous activity, piloerection. 71 h as well as 94 h, 99 h post-dose: piloerection 117 h until the end of the observation period: no further symptoms were observed.

Animal No. 3 of step 1 (2000 mg/kg bw): 30 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 2 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, salivation. 4 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation. 28 h post-dose: this animal was found dead.

Animal no. 1 of step 2 (2000 mg/kg bw):1 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, stertorousness. 3 h as well as 22 h, 27 h post-dose: piloerection. 45 h until the end of the observation period: no further symptoms were observed.

Animal no. 2 of step 2 (2000 mg/kg bw): 1 h post-dose: slightly reduced spontaneous activity, apathy, piloerection. 3 h as well as 22 h, 27 h, 45 h post-dose: piloerection. 69 h until the end of the observation period: no further symptoms were observed.

Animal no.3 of step 2 (2000 mg/kg bw): 1 h post-dose: piloerection. 4 h as well as 23 h, 28 h, 46 h post-dose: piloerection. 69 h until the end of the observation period: no further symptoms were observed.

Absolute body weights in g :

Animal No.	Sex	Day 0	Day 7	Day 14
Step 1 (2000 mg/kg bw)
1	female	180	195	220
2	female	173	154	193
3	female	172	157-this animal was found dead.
Step 2 (2000 mg/kg bw)
1	female	177	158	195
2	female	177	178	197
3	female	176	184	190

Interpretation of results:

harmful

Remarks:

Migrated information criteria given in Annex VI of the Directive 2001/59/EC Criteria used for interpretation of results: EU

Conclusions:

Considering the reported data of this toxicity test it can be stated that the test item N-(Hydrogenated tallow)-1,3-diaminopropane showed oral toxic
characteristics.
According to GHS (Globally Harmonized Classification System) the test item N-(Hydrogenated tallow)-1,3-diaminopropane was classified into Category 5.
(LD50 cutoff:300 mg/kg bw).

Executive summary:

The acute toxic class method for oral toxicity was performed on nulliparous female HsdRccHan:WIST rats, who received a single exposure of N-(Hydrogenated tallow)-1,3-diaminopropane via oral gavage.

A careful clinical examination was made several times on the day of dosing. Parts of this were at least three observations within the first four hours post-dose. Animals were observed once a day thereafter for 14 days. Cage side observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross necropsy. All gross pathological changes were recorded.

In step 1 three female rat were dosed by oral gavage with 2000 mg N-(Hydrogenated tallow)-1,3-diaminopropane/ kg body weight in cotton seed oil. One of the animals died 28 h post-dose. Signs of slight toxicity indicated by apathy, piloerection and reduced spontaneous activity were observed during the first 3 and 5 days in animal 1 and 2 of step 1, respectively. No further symptoms were observed in these two animals until the end of the observation period. In animal 3 apathy, piloerection, reduced spontaneous activity and salvation were evident after dosing and the animal was found dead 28 h post-dose. In step 2 three additional female rats were dosed with 2000 mg N-(Hydrogenated tallow)-1,3-diaminopropane/ kg body weight. No compound related mortality was recorded for any of these animals. Observed signs of toxicity were reduced spontaneous activity, apathy, pilerection and stertorousness. All animals of step 2 recovered within 3 days. According to the toxic class regime no further testing was required.

Following pathological changes were observed in animal 3 of step 1 (2000 mg/kg bw): The dead animal was found in a lateral position. The stomach as well as small and large intestine were at least partly filled with solid content. Following pathological changes were observed in animal 2 of step 2 (2000 mg/kg bw): The spleen was enlarged, with a weight of 0.872 g.

Considering the reported data of this toxicity test it can be stated that the test item N-(Hydrogenated tallow)-1,3-diaminopropane showed oral toxic characteristics. On basis of the test results and in conformity with the criteria given in Annex VI of the Directive 2001/59/EC the substance should be classified as harmful. According to GHS (Globally Harmonized Classification System) the test item N-(Hydrogenated tallow)-1,3-diaminopropane was classified into Category 5 (LD₅₀cut-off: 2500 mg/kg bw).

The acute oral toxicity test was conducted under GLP according to the guidelines for the acute toxic class method OECD 423, EU method B1.tris and OPPTS 870.110 without deviation.

Reference 13

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007-11-27 to 2008-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: HsdRccHan:WIST (Full-Barrier)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Weight at study initiation:
Step 1: 176-194 g
Step 2: 172-184 g
Step 3: 158-176 g

- Fasting period before study: overnight
- Housing: Macrolon cages on Altromin saw fiber bedding
- Diet: ad libitum, Altromin 1324 (TPF)
- Water: ad libitum (drinking water)
- Acclimation period: at least 5 days
- other: SPF animals, marked individually by tail painting

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
Step 1: 1 g/ 5 mL and 2 g/10 mL cotton seed oil
Step 2: 0.3 g/ 10 mL cotton seed oil
Step 3: 0.3 g/ 10 mL cotton seed oil
- Amount of vehicle: 10 mL/ kg body weight
- Justification for choice of vehicle: non-toxic characteristic
- Lot/batch no.: Lot 067K0116, sigma Chemicals

MAXIMUM DOSE VOLUME APPLIED: 1.94 mL

CLASS METHOD
- Rationale for the selection of the starting dose: 2000 mg/ kg was selected as the starting dose

Doses:

Step 1: 2000 mg/ kg
Step 2 and 3: 300 mg/ kg body weight

No. of animals per sex per dose:

female nulliparous rats
2000 mg/ kg dose (Step 1): 3 animals
300 mg/ kg dose (Step 2 and 3): 3 animals + 3 animals

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: A careful clinical examination was made several times on the day of dosing.
Parts of this were at least three observations within the first four hours post-dose.
Animals were observed once a day thereafter.
The animals were weighed prior to the administration and once a week thereafter.

- Necropsy of survivors performed: yes

- Other examinations performed: Cage side observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity
and behaviour pattern were examined. Particular attention was directed to observations of tremor,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

not applicable

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

1 000 mg/kg bw

Remarks on result:

other: acute toxic class method; according to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4

Mortality:

Step 1 (2000 mg/kg bw): 2 animals were found dead, one after 17.5 h and one after 76.25 h
Step 2 (300 mg/kg bw): no mortality observed
Step 3 (300 mg/kg bw): no mortality observed

Clinical signs:

other: see remarks on results including tables and figures

Gross pathology:

Animal no. 1 of step 1 (2000 mg/ kg bw):
The dead animal was found in a lateral position. Small and large intestine had
liquid content.
Animal no. 2 of step 1 (2000 mg/ kg bw):
The animal was found in a face down position. The anus was full of faeces.
The stomach contained rests of the test item and was bloated.

Signs of toxicity related to dose level used, time of onset and duration:

Animal no. 1 of step 1(2000 mg/kg bw): 05 min post-dose: salivation. 3 h 45 min post-dose: apathy, piloerection. 20 h 45 min as well as 26 h 15 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 44 h 45 min post-dose: slightly reduced spontaneous activity, apathy, piloerection, diarrhea. 48 h 45 min post-dose: slightly reduced spontaneous activity, apathy, piloerection, diarrhea, weight loss. 76 h 15 min post-dose: the animal was found dead.

Animal no. 2 of step 1(2000 mg/kg bw): 30 min post-dose: slightly reduced spontaneous activity, piloerection. 17 h 30 min post-dose: the animal was found dead.  

Animal no. 3 of step 1 (2000 mg/kg bw): 30 min post-dose: slightly reduced spontaneous activity, piloerection, salivation. 17 h 30 min as well as 41 h 15 min, 48 h 30 min post-dose: moderately reduced spontaneous activity, piloerection, diarrhea, stertorousness. 66 h post-dose: moderately reduced spontaneous activity, piloerection, diarrhea. 93 h 30 min as well as 114 h, 138 h post-dose: slightly reduced spontaneous activity, piloerection, respiratory sounds, diarrhea. 166 h as well as 191 h post-dose: slightly reduced spontaneous activity, piloerection 217 h as well as 238 h 15 min 257 h post-dose: piloerection. 12 days post-dose until the end of the observation period: no further symptoms were observed.  

Animal No. 1 of step 2 (300 mg/kg bw 45 min as well as 21 h 45 min post-dose: slightly reduced spontaneous activity, piloerection. 2 h 45 min post-dose: piloerection, half-eyelid closure. 26 h 45 min as well as 44 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, bloody snout. 68 h 45 min as well as 92 h 45 min post-dose: piloerection. 119 h 45 min post-dose: piloerection, chromodakryorrhoe. 145 h 15 min post-dose: piloerection, chromodakryorrhoe, heavy respiration, articulation, convulsion. 164 h 45 min as well as 189 h 45 min, 213 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, stertorousness, weight loss. 236 h 45 min as well as 260 h 45 min, 284 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, stertorousness. 13 days until the end of the observation period: no further symptoms were observed.

Animal no. 2 of step 2 (300 mg/kg bw): 45 min as well as 2 h 45 min, 21 h 45 min, 26 h 15 min, 44 h 45 min postdose: slightly reduced spontaneous activity, piloerection. 68 h 45 min as well as 92 h 45 min post-dose: piloerection. 5 days until the end of the observation period: no further symptoms were observed.

Animal no.3 of step 2 (300 mg/kg bw): 45 min as well as 2 h 45, 21 h 45, 26 h 45 min, 44 h 45 post-dose: slightly reduced spontaneous activity, piloerection. 68 h 45 min as well as 92 h 45 min post-dose: piloerection. 119 h 45 min post-dose: piloerection, chromodakryorrhoe. 145 h 15 min post-dose: piloerection, chromodakryorrhoe, heavy respiration, articulation, convulsion. 7 days post-dose until the end of the observation period: no further symptoms were observed.

Animal no. 1,2 and 3 of step 3 (300 mg/kg bw 30 min as well as 2 h 30 min post-dose: slightly reduced spontaneous activity, piloerection. 19 h 30 min as well as 43 h 30 min, 67 h 30 min, 94 h 30 min post-dose: piloerection. 5 days post-dose until the end of the observation period: no further symptoms were observed.  

Absolute body weights in g

Animal No.	Sex	Day 0	Day 7	Day 14
Step 1 (2000 mg/kg bw)
1	female	177	149- This animal was found dead.
2	female	194	185- This animal was found dead.	.
3	female	176	138	178
Step 2 (300 mg/kg bw)
1	female	184	145	150
2	female	172	193	200
3	female	172	162	188
Step 3 (300 mg/kg bw)
1	female	173	190	203
2	female	158	179	187
3	female	176	198	211

Interpretation of results:

harmful

Remarks:

Migrated information criteria given in Annex VI of the Directive 2001/59/EC Criteria used for interpretation of results: EU

Conclusions:

Considering the reported data of this toxicity test it can be stated that the test item N-Oleyl-1,3-diaminopropane showed acute oral toxic characteristics. According to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4 (LD50
cut-off: 1000 mg/kg bw).

Executive summary:

The acute toxic class method for oral toxicity was performed on nulliparous female HsdRccHan:WIST rats, who received a single exposure of N-Oleyl-1,3-diaminopropane (liquid/paste) via oral gavage.

A careful clinical examination was made several times on the day of dosing. Parts of this were at least three observations within the first four hours post-dose. Animals were observed once a day thereafter for 14 days. Cage side observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross necropsy. All gross pathological changes were recorded.

In step 1 three female rats were dosed by oral gavage with 2000 mg N-Oleyl-1,3-diaminopropane/ kg body weight in cotton seed oil. Severe toxicity indicated by salivation, apathy, piloerection, reduced spontaneous activity, diarrhoea and weight loss was observed in this group after the application. Animal 1 and 2 died within 17.5 h and 76.25 h, respectively. Animal 3 recovered on day 12 and survived the 14 days observation period. In step 2 three female rats were dosed with 300 mg N-Oleyl-1,3-diaminopropane/ kg body weight. Animal 1 of step 2 showed signs of toxicity indicated by reduced spontaneous activity, half eyelid-closure, piloerection, bloody snout, chromodakryorrhoe, heavy respiration, articulation, convulsion, weight loss and stertorousness and recovered on day 13. Animal 2 of step 2 displayed reduced spontaneous activity and piloerection and recovered on day 5. Animal 3 of step 2 showed following signs of toxicity: piloerection, reduced spontaneous activity, chromodakryorrhoe, heavy respiration, articulation and convulsion. No further symptoms were observed from day 7 on. In step 3 three additional female rats were dosed with 300 mg N-Oleyl-1,3 -diaminopropane/ kg body weight. All three animals showed signs of toxicity indicated by reduced spontaneous activity and piloerection. No more symptoms were observed from day 5 until the end of the observation period. According to the toxic class regime no further testing was required.

Following pathological changes were observed. Animal 1 of step 1 (2000 mg/kg bw) was found dead in a lateral position after 3 days and its small and large intestine had liquid content. Animal 2 of step 1 (2000 mg/kg bw) was found dead in a face down position after 17.5 h. Its anus was full of faeces and the stomach contained rests of the test item and was bloated.

Considering the reported data of this toxicity test it can be stated that the test item N-Oleyl-1,3-diaminopropane showed acute oral toxic characteristics. On basis of the test results and in conformity with the criteria given in Annex VI of the Directive 2001/59/EC the substance should be classified as harmful. According to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4 (LD₅₀ cut-off: 1000 mg/kg bw).

The acute oral toxicity test was conducted under GLP according to the guidelines for the acute toxic class method OECD 423, EU method B1.tris and OPPTS 870.110 without deviation.

Reference 14

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007-11-27 to 2008-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: HsdRccHan: WIST (SPF)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Weight at study initiation:
Step 1: 168 g (1 animal)
Step 2: 189-200 g
Step 3: 133-173 g
Step 4: 180-214 g

- Fasting period before study: overnight and 3-4 hours after dosing
- Housing: macrolon cages on Altromin saw fibre bedding
- Diet: ad libitum (Altromin 1324 (TPF)
- Water: ad libitum (drinking water)
- Acclimation period: at least 5 days
- Other: marked individually by tail painting, SPF animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
Step 1: 1.0 g/ 5 mL
Step 2: 0.3 g/ 10 mL
Step 3: 0.05 g/ 10 mL
Step 4: 0.05 g/ 10 mL

- Amount of vehicle: 10 mL/ kg body weight
- Justification for choice of vehicle: non-toxic and lipophil
- Lot/batch no. : Lot 067K0116, Sigma Chemicals

MAXIMUM DOSE VOLUME APPLIED: 2.00 mL

DOSAGE PREPARATION : freshly mixed to homogeneity prior to administration

CLASS METHOD
- Rationale for the selection of the starting dose:
preliminary test with one animal performed (2000 mg/kg), due to immeadiate preacute lethality
the starting dose was choosen to be 300 mg/kg bw

Doses:

Step 1: 2000 mg/kg bw
Step 2: 300 mg/kg bw
Step 3: 50 mg/kg bw
Step 4: 50 mg/kg bw

No. of animals per sex per dose:

Step 1 (2000 mg/kg bw): 1 female rat
Step 2 (300 mg/kg bw): 3 female rats
Step 3 (50 mg/kg bw): 3 female rats
Step 4 (50 mg/kg bw): 3 female rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: A careful clinical examination was made several times on the day of dosing.
Parts of this were at least three observations within the first four hours post-dose.
Animals were observed once a day thereafter.
The animals were weighed prior to the administration and once a week thereafter.

- Necropsy of survivors performed: yes

- Other examinations performed: Cage side observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity
and behaviour pattern were examined. Particular attention was directed to observations of tremor,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

not applicable

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

200 mg/kg bw

Remarks on result:

other: acute toxic class method; according to GHS (Globally Harmonized Classification System) the test item N-C12,14 alkyl-1,3-diaminopropane was classified into Category 3

Mortality:

Step 1 (2000 mg/kg bw, 1 female rat): within 1 h 30 min post-dose the animal was found dead
Step 2 (300 mg/kg bw): within 2 days post-dose all three animals were found dead
Step 3 (50 mg/kg bw): no deaths observed within the observation period
Step 4 (50 mg/kg bw): 1 animal was found dead 13 days post-dose

Clinical signs:

other: see remarks on results including tables and figures

Gross pathology:

Animal no. 1 of step 1 (2000 mg/ kg bw):
The stomach, small and large intestine were bloody.
Animal no.1 of step 2 (300 mg/ kg bw):
The stomach was bloody. The stomach and small intestines had a yellow content. The liver had a
dark colour.
Animal no.2 of step 2 (300 mg/ kg bw):
The animal was found in a face down position. The stomach mucosa was bloody and the stomach
content was yellow. The anus was smeared with faeces.
Animal no.3 of step 2 (300 mg/ kg bw):
The animal was found in a face down position. The stomach mucosa was
bloody and the stomach content was yellow.
Animal no. 2 of step 4 (50 mg/ kg bw):
Small and large intestine and the stomach were bloated. The spleen weighed 150 g.

Signs of toxicity related to dose level used, time of onset and duration:

Animal no. 1 of step 1(2000 mg/kg bw): 05 min post-dose: apathy, salivation. 20 min post-dose: moderately reduced spontaneous activity, salivation, diarrhoea, face down position. 1 h 30 min post-dose: this animal was found dead.

Animal no. 1 of step 2(300 mg/kg bw): 1 h 30 min post-dose: slightly reduced spontaneous activity, piloerection. 3 h 30 min post-dose: moderately reduced spontaneous activity, piloerection, diarrhoea. 22 h 30 min post-dose: moderately reduced spontaneous activity, piloerection, diarrhoea, sunken flank. 26 h 30 min: this animal was found dead.

Animal no. 2 of step 2 (300 mg/kg bw): 30 min as well as 3 h post-dose: slightly reduced spontaneous activity, apathy, piloerection. 24 h as well as 45 h post-dose: slightly reduced spontaneous activity, piloerection. 71 h as well as 94 h, 99 h post-dose: piloerection 117 h until the end of the observation period: no further symptoms were observed.

Animal No. 3 of step 2 (300 mg/kg bw): 45 min post-dose: slightly reduced spontaneous activity, piloerection, diarrhoea. 17 h 45 min post-dose: moderately reduced spontaneous activity, piloerection, diarrhoea. 41 h 15 min post-dose: this animal was found dead.

Animal no. 1 of step 3 (50 mg/kg bw): 45 min post-dose: slightly reduced spontaneous activity, piloerection. 17 h 45 min post-dose: moderately reduced spontaneous activity, piloerection, diarrhoea. 41 h 15 min post-dose: this animal was found dead.

Animal no. 2 of step 3 (50 mg/kg bw): 1 h, 20 h, 44 h post-dose: slightly reduced spontaneous activity, piloerection. 48 h post-dose: moderately reduced spontaneous activity, piloerection. 68 h, 92 h, 116 h post-dose: slightly reduced spontaneous activity, piloerection. 6 days post-dose until the end of the observation period: no further symptoms were observed.

Animal no.3 of step 3 (50 mg/kg bw): 2 h 30 min as well as 21 h 30 min, 46 h 30 min post-dose: piloerection. 4 h post-dose: slightly reduced spontaneous activity, piloerection. 3 days post-dose until the end of the observation period: no further symptoms were observed.

Animal no. 1 of step 4 (50 mg/kg bw): 3 h post-dose: slightly reduced spontaneous activity, piloerection. 21 h post-dose: slightly reduced spontaneous activity, piloerection, diarrhea. 45 h 30 min post-dose: slightly reduced spontaneous activity, piloerection. 69 h 30 min post-dose: piloerection. 97 h post-dose: slight piloerection, respiratory sounds. 119 h post-dose: piloerection. 6 days until the end of the observation period: no further symptoms were observed.

Animal no. 2 of step 4 (50 mg/kg bw): 3 h post-dose: slightly reduced spontaneous activity, piloerection. 21 h post-dose: slightly reduced spontaneous activity, piloerection, diarrhea. 45 h 30 min post-dose: slightly reduced spontaneous activity, piloerection. 69 h 30 min post-dose: piloerection. 93 h 30 min as well as 119 h post-dose: piloerection. 142 h 30 min post-dose: no symptoms observed. 11 days post-dose: slightly reduced spontaneous activity, piloerection. 12 days post-dose: slightly reduced spontaneous activity, piloerection, respiratory sounds. 13 days post-dose: this animal was found dead.

Animal no.3 of step 4 (50 mg/kg bw): 3 h as well as 69 h 30 min, 93 h 30 min, 119 h post-dose: piloerection. 3 h 30 min as well as 45 h 30 min: slightly reduced spontaneous activity, piloerection. 21 h post-dose: slightly reduced spontaneous activity, piloerection, diarrhea. 142 h 30 min post-dose until the end of the observation period: no further symptoms were observed.

Absolute body weights in g :

Animal No.	Sex	Day 0	Day 7	Day 14
Step 1 (2000 mg/kg bw)
1	female	168	This animal was found dead.
Step 2 (300 mg/kg bw)
1	female	189	This animal was found dead.
2	female	200	188- This animal was found dead.	.
3	female	199	192- This animal was found dead.
Step 3 (50 mg/kg bw)
1	female	173	205	206
2	female	133	162	177
3	female	146	163	174
Step 4 (50 mg/kg bw)
1	female	206	200	196
2	female	180	182	This animal was found dead.
3	female	217	252	255

Interpretation of results:

toxic

Remarks:

Migrated information criteria given in Annex VI of the Directive 2001/59/EC Criteria used for interpretation of results: EU

Conclusions:

Considering the reported data of this toxicity test it can be stated that the test item N-C12,14 alkyl-1,3-diaminopropane showed acute oral toxic
characteristics.
According to GHS (Globally Harmonized Classification System) the test item N-C12,14 alkyl-1,3-diaminopropane was classified into Category 3
(LD50 cut-off: 200 mg/kg bw).

Executive summary:

The acute toxic class method for oral toxicity was performed on nulliparous female HsdRccHan:WIST rats, who received a single exposure of N-C12,14 alkyl-1,3-diaminopropane via oral gavage.

A careful clinical examination was made several times on the day of dosing. Parts of this were at least three observations within the first four hours post-dose. Animals were observed once a day thereafter for 14 days. Cage side observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross necropsy. All gross pathological changes were recorded.

In step 1 single female rat was dosed by oral gavage with 2000 mg N-C12,14 alkyl-1,3-diaminopropane/ kg body weight in cotton seed oil. Severe, immediate toxicity indicated by salivation, face down position, diarrhoea and moderately reduced spontaneous activity was observed in this animal, followed by death at 1 h and 30 min post-dose. In step 2 three female rats were dosed with 300 mg N-C12,14 alkyl-1,3-diaminopropane/ kg body weight. All three animals showed severe signs of toxicity indicated by piloerection, sunken flank, diarrhoea and reduced spontaneous activity and died within 2 days post-dose. In step 3 three female rats were dosed with 50 mg N-C12,14 alkyl-1,3-diaminopropane/ kg body weight. No mortality was observed in all three animals.

Observed signs of toxicity were reduced spontaneous activity, piloerection and diarrhoea. The animals had recovered within 3 (animal 2) to 6 days (animal 1 and 3) post-dose and showed no symptoms thereafter. In step 4 three additional female rats were dosed with 50 mg N-C12,14 alkyl-1,3-diaminopropane/ kg body weight. Mortality was observed in one animal. Observed signs of toxicity were reduced spontaneous activity, piloerection and diarrhoea. All three animals recovered within 6 days post-dose. Animal 1 and 3 of step 4 showed no further symptoms thereafter. Animal 2 of step 4 showed the same toxicity signs again from day 11 on and was found dead on day 13. According to the toxic class regime no further testing was required.

Following pathological changes were observed in the step 1 animal (2000 mg/kg bw): The stomach, small and large intestine were bloody. Animal 1 of step 2 (300 mg/kg bw) exhibited a bloody stomach. The stomach and small intestines had a yellow content. The liver had a dark colour. Animal 2 of step 2 (300 mg/kg bw) was found in a face down position and exhibited a bloody stomach mucosa and the stomach content was yellow. The anus was smeared with faeces. Animal 3 of step 2 (300 mg/kg bw) was found in a face down position and exhibited a bloody stomach mucosa and a yellow stomach content. In animal 2 of step 4 (50 mg/kg bw) the small and large intestine and the stomach were bloated. The spleen weight was markedly increased (150 g).

Considering the reported data of this toxicity test it can be stated that the test item N-C12,14 alkyl-1,3-diaminopropane showed acute oral toxic characteristics. On basis of the test results and in conformity with the criteria given in Annex VI of the Directive 2001/59/EC the substance should be classified as toxic. According to GHS (Globally Harmonized Classification System) the test item N-C12,14 alkyl-1,3-diaminopropane was classified into Category 3 (LD₅₀ cut-off: 200 mg/kg bw).

The acute oral toxicity test was conducted under GLP according to the guidelines for the acute toxic class method OECD 423, EU method B1.tris and OPPTS 870.110 without deviation.

Reference 15

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-11-16 to 2008-01-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: HsdRccHan:WIST (Full-Barrier)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Weight at study initiation:
Step 1: 154-166 g
Step 2: 171-191 g
Step 3: 172-179 g

- Fasting period before study: overnight
- Housing: Macrolon cages on Altromin saw fiber bedding
- Diet: ad libitum, Altromin 1324
- Water: ad libitum (drinking water)
- Acclimation period: at least 5 days
-Other: marked individually by tail painting, SPF animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
Step 1: 1 g/ 5 mL and 2 g/10 mL cotton seed oil
Step 2: 0.3 g/ 10 mL cotton seed oil
Step 3: 0.3 g/ 10 mL cotton seed oil
- Amount of vehicle: 10 mL/ kg body weight
- Justification for choice of vehicle: non-toxic characteristic
- Lot/batch no.: Lot 067K0116, Sigma Chemicals

MAXIMUM DOSE VOLUME APPLIED: 1.91 mL

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: 2000 mg/ kg was selected as the starting dose

Doses:

Step 1: 2000 mg/ kg
Step 2 and 3: 300 mg/ kg body weight

No. of animals per sex per dose:

female nulliparous rats
2000 mg/ kg dose (Step 1): 3 animals
300 mg/ kg dose (Step 2 and 3): 3 animals + 3 animals

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing:
A careful clinical examination was made several times on the day of dosing.
Parts of this were at least three observations within the first four hours postdose.
Animals were observed once a day thereafter. The animals were weighed prior to
the administration and once a week thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed:
Cage side observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity
and behaviour pattern were examined. Particular attention was directed to observations of tremor,
convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period
the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross
necropsy. All gross pathological changes were recorded.

Statistics:

not applicable

Preliminary study:

no data

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

500 mg/kg bw

Remarks on result:

other: acute toxic class method; according to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4

Mortality:

Step 1 (2000 mg/kg bw): all 3 animals died within 24 h (one was euthanised due to animal welfare reasons, two were found dead in their cages)
Step 2 (300 mg/kg bw): 1 animal was found dead 10 days after treatment
Step 3 (300 mg/kg bw): no mortality observed

Clinical signs:

other: see remarks on results including tables and figures

Gross pathology:

Effect on organs (related to dose level):

Animal no. 1 of step 1 (2000 mg/ kg bw):
The animal was found dead in a lateral position. Rests of the test item were
found in the stomach. The lungs were infiltrated with blood.

Animal no. 2 of step 1 (2000 mg/ kg bw):
Rests of the test item were found in the stomach. The lungs were infiltrated
with blood.

Animal no. 3 of step 1 (2000 mg/ kg bw):
Rests of the test item were found in the stomach, which was bloated. The
small intestine was empty and of a yellow colour. This animal was
euthanised due to animal welfare reasons.

Animal no. 2 of step 2 (300 mg/ kg bw):
Stomach, small and large intestine bloated and empty.

Signs of toxicity related to dose level used, time of onset and duration:

Animal no. 1 of step 1 (2000 mg/kg bw): 30 min post-dose: salivation. 2 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure. 4 h post-dose: moderately reduced spontaneous activity, piloerection, salivation, full eyelid-closure. 5 h as well as 9 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure. 24 h post-dose the animal was found dead.

Animal no. 2 of step 1 (2000 mg/kg bw): 30 min post-dose: salivation. 2 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure. 4 h post-dose: moderately reduced spontaneous activity, piloerection, salivation, full eyelid-closure. 5 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure. 9 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure, slight articulation. 24 h post-dose the animal was found dead.

Animal No. 3 of step 1 (2000 mg/kg bw): 30 min post-dose: salivation. 2 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, salivation. 4 h post-dose: moderately reduced spontaneous activity, piloerection, salivation, full eyelid-closure. 5 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure. 9 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation, half eyelid-closure, slight articulation. 24 h post-dose: moderately reduced spontaneous activity, piloerection, salivation, half eyelid-closure, severe articulation. 25 h post-dose: for animal welfare reasons this animal was euthanised.

Animal no. 1 of step 2 (300 mg/kg bw): 3 h 25 min as well as 7 h, 24 h, 30 h post-dose: piloerection. 48 h post-dose until the end of the observation period: no symptoms were observed.

Animal no. 2 of step 2 (300 mg/kg bw): 45 min as well as 2 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, half eyelid-closure. 4 h 45 min post-dose: slightly reduced spontaneous activity, piloerection. 23 h 45 min post-dose: weight loss, stertorousness. 47 h 45 min as well as 71 h 45 min, 95 h 45 min, 119 h 45 min, 143 h 45 min, 167 h 45 min, 191 h 45 min post-dose: piloerection, weight loss, stertorousness. 215 h 45 min. post-dose: moderately reduced spontaneous activity, piloerection, weight loss, stertorousness. 10 days (238 h 45 min) post-dose: the animal was found dead.

Animal no. 3 of step 2 (300 mg/kg bw): 1 h 15 min post-dose: piloerection. 2 h 45 min as well as 4 h 45 min post-dose: slightly reduced spontaneous activity, piloerection. 23 h 45 min post-dose until the end of the observation period: no symptoms were observed.

Animal no. 1, 2 and 3 of step 3 (300 mg/kg bw): 15 min as well as 1 h 45 min post-dose: slightly reduced spontaneous activity, piloerection. 3 h 45 min post-dose: moderately reduced spontaneous activity, piloerection. 21 h 45 min as well as 45 h 45 min post-dose: slightly reduced spontaneous activity, piloerection. 69 h 45 min post-dose: piloerection. 93 h 45 min post-dose until the end of the observation period: no symptoms were observed.

Absolute body weights in g

Animal No.	Sex	Day 0	Day 7	Day 14
Step 1 (2000 mg/kg bw)
1	female	154	This animal was found dead.
2	female	157	This animal was found dead.
3	female	166	This animal was found dead
Step 2 (300 mg/kg bw)
1	female	171	189	212
2	female	191	137	This animal was found dead.
3	female	173	187	200
Step 3 (300 mg/kg bw)
1	female	172	180	196
2	female	179	192	206
3	female	173	165	182

Interpretation of results:

harmful

Remarks:

Migrated information criteria given in Annex VI of the Directive 2001/59/EC Criteria used for interpretation of results: EU

Conclusions:

Considering the reported data of this toxicity test it can be stated that the test item N-Oleyl-1,3-diaminopropane showed acute oral toxic characteristics. According to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4 (LD50
cut-off: 500 mg/kg bw).

Executive summary:

The acute toxic class method for oral toxicity was performed on nulliparous female HsdRccHan:WIST rats, who received a single exposure of N-Oleyl-1,3-diaminopropane (liquid) via oral gavage.

A careful clinical examination was made several times on the day of dosing. Parts of this were at least three observations within the first four hours post-dose. Animals were observed once a day thereafter for 14 days. Cage side observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross necropsy. All gross pathological changes were recorded.

In step 1 three female rats were dosed by oral gavage with 2000 mg N-Oleyl-1,3-diaminopropane/ kg body weight in cotton seed oil. Severe toxicity indicated by salivation, apathy, piloerection and half eyelid-closure were observed in this group after the application. Two animals died within 24 h and one was euthanized 25 h post-dose for animal welfare reasons. In step 2 three female rats were dosed with 300 mg N-Oleyl-1,3-diaminopropane/ kg body weight. Two animals showed slight signs of toxicity indicated by piloerection and reduced spontaneous activity until 48 h post-dose and had recovered completely thereafter. One animal showed severe signs of toxicity indicated by reduced spontaneous activity, half eyelid-closure, piloerection, weight loss and stertorousness and was found dead after 10 days. Therefore three additional female rats were dosed with 300 mg N-Oleyl-1,3 -diaminopropane/kg body weight. Observed signs of toxicity were slightly reduced spontaneous activity and piloerection until 4 days post-dose. The animals showed no symptoms thereafter. According to the toxic class regime no further testing was required.

Following pathological changes were observed in the step 1 animals (2000 mg/kg bw): rests of the item were found in the stomach of all three rats, the lungs were infiltrated with blood in all three rats, one animal had a bloated stomach and an empty small intestine of a yellow colour. The animal of step 2 (300 mg/kg bw) that was found dead after 10 days had a bloated and empty stomach and small and large intestine.

Considering the reported data of this toxicity test it can be stated that the test item N-Oleyl-1,3-diaminopropane showed acute oral toxic characteristics. On basis of the test results and in conformity with the criteria given in Annex VI of the Directive 2001/59/EC the substance should be classified as harmful. According to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4 (LD₅₀ cut-off: 500 mg/kg bw).

The acute oral toxicity test was conducted under GLP according to the guidelines for the acute toxic class method OECD 423, EU method B1.tris and OPPTS 870.110 without deviation.

Reference 16

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2007-11-21 to 2008-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: HsdRccHan: WIST (Full-Barrier)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Weight at study initiation:
Step 1: 168 g (1 animal)
Step 2: 170-186 g
Step 3: 141-177 g
Step 4: 135-158 g
Step 5: 128-131 g

- Fasting period before study: overnight and 3-4 hours after dosing
- Housing: macrolon cages on Altromin saw fibre bedding
- Diet: ad libitum (Altromin 1324 (TPF)
- Water: ad libitum (drinking water)
- Acclimation period: at least 5 days
- Other: marked individually by tail painting, SPF animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
Step 1: 1.0 g/ 5 mL
Step 2: 0.3 g/ 10 mL
Step 3: 0.3 g/ 10 mL
Step 4: 0.1 g/ 20 mL
Step 5: 0.1 g/ 20 mL
- Amount of vehicle: 10 mL/ kg body weight
- Justification for choice of vehicle: non-toxic and lipophil
- Lot/batch no. : Lot 067K0116, Sigma Chemicals

MAXIMUM DOSE VOLUME APPLIED: 1.86 mL

DOSAGE PREPARATION : freshly mixed prior to administration

CLASS METHOD
- Rationale for the selection of the starting dose:
preliminary test with one animal performed (2000 mg/kg), due to immeadiate preacute lethality
the starting dose was choosen to be 300 mg/kg bw

Doses:

Step 1: 2000 mg/kg bw
Step 2: 300 mg/kg bw
Step 3: 300 mg/kg bw
Step 4: 50 mg/kg bw
Step 5: 50 mg/kg bw

No. of animals per sex per dose:

Step 1 (2000 mg/kg bw): 1 female rat
Step 2 (300 mg/kg bw): 3 female rats
Step 3 (300 mg/kg bw): 3 female rats
Step 4 (50 mg/kg bw): 3 female rats
Step 5 (50 mg/kg bw): 3 female rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: A careful clinical examination was made several times on the day of dosing.
Parts of this were at least three observations within the first four hours post-dose.
Animals were observed once a day thereafter.
The animals were weighed prior to the administration and once a week thereafter.

- Necropsy of survivors performed: yes

- Other examinations performed: Cage side observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity
and behaviour pattern were examined. Particular attention was directed to observations of tremor,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

not applicable

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

300 mg/kg bw

Remarks on result:

other: acute toxic class method; according to GHS (Globally Harmonized Classification System) the test item N-coco-1,3-diaminopropane was classified into Category 3

Mortality:

Step 1 (2000 mg/kg bw, 1 female rat): within 1 h 30 min post-dose the animal was found dead
Step 2 (300 mg/kg bw): no deaths observed within the observation period
Step 3 (300 mg/kg bw): Animal 2 was found dead five days and animal 3 three days post-dose
Step 4 and 5 ( 50 mg/kg bw): no deaths observed within the observation period

Clinical signs:

other: see remarks on results including tables and figures

Gross pathology:

Animal no. 1 of step 1 (2000 mg/ kg bw):
The stomach showed haemorrhage with a foamy content. The small intestine
also showed haemorrhage with a bloody content. The large intestine and appendix were bloody.
Animal no.2 of step 3 (300 mg/ kg bw):
The animal was found dead in a lateral position and exhibited piloerection.The anus was smeared with
faeces.
Animal no. 3 of step 3 (300 mg/ kg bw):
The stomach was bloody. The small intestine was slightly bloody.

Signs of toxicity related to dose level used, time of onset and duration:

Animal no. 1 of step 1 (2000 mg/kg bw): 30 min post-dose: moderately reduced spontaneous activity, salivation, face down position.1 h 30 min post-dose: this animal was found dead.

Animal no. 1 of step 2 (300 mg/kg bw): 25 min post-dose: slightly reduced spontaneous activity, apathy, piloerection.1 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, half eyelid-closure. 5 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, sunken flank. 22 h 45 min as well as 28 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, sunken flank, diarrhoea. 46 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, diarrhoea, bloody snout, bloody mouth. 50 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, diarrhoea, weight loss. 78 h 15 min post-dose: piloerection, diarrhoea, weight loss. 98 h 10 min post-dose: slightly reduced spontaneous activity, piloerection, diarrhoea, heavy breathing (rattling). 119 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, apathy, weight loss. 143 h 45 min post-dose: apathy, piloerection.166 h 45 min post-dose: weight gain.

Animal No. 2 and 3 of step 2 (300 mg/kg bw): 30 min post-dose: slightly reduced spontaneous activity, apathy, salivation. 2 h 15 min post-dose: slightly reduced spontaneous activity (animal no. 3), moderately reduced spontaneous activity (animal no. 2), piloerection, half eyelid-closure. 4 h 15 min post-dose: moderately reduced spontaneous activity, piloerection. 23 h 15 min post-dose: slightly reduced spontaneous activity, piloerection. 28 h 15 min post-dose: moderately reduced spontaneous activity, piloerection, half eyelid-closure. 2 days post-dose until the end of the observation period: no further symptoms were observed.

Animal no. 1 of step 3 (300 mg/kg bw): 15 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 2 h as well as 4 h 30 min, 22 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure. 47 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure, diarrhoea, bloody snout, anal discharge. 71 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, diarrhoea, weight loss. 97 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, diarrhoea, weight loss. 121 h post-dose: slightly reduced spontaneous activity, piloerection, diarrhoea. 144 h post-dose: slightly reduced spontaneous activity, piloerection. 7 days post-dose until the end of the observation period: no further symptoms were observed.

Animal no. 2 of step 3 (300 mg/kg bw): 15 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 2 h as well as 4 h, 22 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure. 47 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure, diarrhoea, anal discharge. 71 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, diarrhoea, weight loss. 97 h post-dose: severely reduced spontaneous activity, piloerection, diarrhoea, weight loss. 5 days post-dose the animal was found dead.

Animal no.3 of step 3 (300 mg/kg bw): 15 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 2 h as well as 4 h, 22 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure. 47 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, half eyelid-closure, diarrhoea, anal discharge, bloody snout.3 days post-dose the animal was found dead.

Animal no. 1, 2 and 3 of step 4 and 5 (50 mg/kg bw): No compound related mortality was recorded for any animal.

Absolute body weights in g :

Animal No.	Sex	Day 0	Day 7	Day 14
Step 1 (2000 mg/kg bw)
1	female	168	This animal was found dead.
Step 2 (300 mg/kg bw)
1	female	170	150	190
2	female	180	189	205
3	female	186	196	213
Step 3 (300 mg/kg bw)
1	female	177	180	201
2	female	141	92-this animal was found dead
3	female	149	This animal was found dead.
Step 4 (50 mg/kg bw)
1	female	135	138	142
2	female	158	191	206
3	female	158	183	197
Step 5 (50 mg/kg bw)
1	female	128	162	177
2	female	131	161	176
3	female	130	163	174

Interpretation of results:

toxic

Remarks:

Migrated information criteria given in Annex VI of the Directive 2001/59/EC Criteria used for interpretation of results: EU

Conclusions:

Considering the reported data of this toxicity test it can be stated that the test item N-Coco-1,3-diaminopropane showed acute oral toxic
characteristics.
According to GHS (Globally Harmonized Classification System) the test item N-Coco-1,3-diaminopropane was classified into Category 3
(LD50 cutoff:300 mg/kg bw).

Executive summary:

The acute toxic class method for oral toxicity was performed on nulliparous female HsdRccHan:WIST rats, who received a single exposure of N-Coco-1,3-diaminopropane via oral gavage.

A careful clinical examination was made several times on the day of dosing. Parts of this were at least three observations within the first four hours post-dose. Animals were observed once a day thereafter for 14 days. Cage side observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross necropsy. All gross pathological changes were recorded.

In step 1 single female rat was dosed by oral gavage with 2000 mg N-coco-1,3-diaminopropane/ kg body weight in cotton seed oil. Severe, immediate toxicity indicated by salivation, face down position and moderately reduced spontaneous activity was observed in this animal, followed by death at 1 h and 30 min post-dose. In step 2 three female rats were dosed with 300 mg N-coco-1,3-diaminopropane/ kg body weight. Animal 1 showed severe signs of toxicity indicated by immediate piloerection followed by half eyelid closure, sunken flank, diarrhoea, bloody snout and mouth, weight loss and heavy breathing . 7 days post-dosing the animal gained weight again and recovered. Animal 2 and 3 showed reduction in spontaneous activity, apathy, salvation, piloerection and half eyelid-closure shortly after dosing. 2 days post-dose until the end of the observation period no further symptoms were evident. In step 3 three female rats were dosed with 300 mg N-coco-1,3 -diaminopropane/ kg body weight. Observed signs of toxicity of animal 1 were reduced spontaneous activity, piloerection, apathy, half eyelid-closure, diarrhoea, bloody snout, anal discharge and weight loss until 7 days post-dose. The animal showed no symptoms thereafter. Animal 2 of step 3 displayed increasing signs of toxicity indicated by reduced spontaneous activity, piloerection, apathy, half eyelid-closure, diarrhoea, anal discharge as well as weight loss and was found dead on day 5 post-dose. Animal 3 of step 3 displayed increasing signs of toxicity indicated by reduced spontaneous activity, piloerection, apathy, half eyelid-closure, diarrhoea, bloody snout, anal discharge and weight loss and was found dead on day 3 post-dose. Animal no. 1, 2 and 3 of step 4 and 5 received 50 mg N-coco-1,3-diaminopropane/ kg body weight by oral gavage. No compound related mortality was recorded for any of the animals.

According to the toxic class regime no further testing was required.

Following pathological changes were observed in the step 1 animal (2000 mg/kg bw): The stomach showed haemorrhage with a foamy content. The small intestine also showed haemorrhage with a bloody content. The large intestine and appendix were bloody. Animal 2 of step 3 (300 mg/kg bw) that was found dead after 5 days in a lateral position exhibited piloerection. Its anus was smeared with faeces. Animal no. 3 of step 3 (300 mg/ kg bw) had a bloody stomach. Its small intestine was slightly bloody.

Considering the reported data of this toxicity test it can be stated that the test item N-coco-1,3-diaminopropane showed acute oral toxic characteristics. On basis of the test results and in conformity with the criteria given in Annex VI of the Directive 2001/59/EC the substance should be classified as toxic. According to GHS (Globally Harmonized Classification System) the test item N-Coco-1,3-diaminopropane was classified into Category 3 (LD₅₀ cut-off: 300 mg/kg bw).

The acute oral toxicity test was conducted under GLP according to the guidelines for the acute toxic class method OECD 423, EU method B1.tris and OPPTS 870.110 without deviation.

Reference 17

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2007-11-27 to 2007-12-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Justification for type of information:

The starting material for this reaction mass is n-tallow alkyltrimethyenediamine CAS No 61791-55-7, which was registered for REACH as N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine(CAS No 1219010-04-4), that is reacted with tall oil fatty acids (CAS No 61790-12-3), 2-ethylhexanoic acid (CAS No 149-57-5) and acetic acid (CAS No 64-18-7). The n-tallow alkyltrimethyenediamine is ca. 52.2% of the reaction mixture with the 2-ethylhexanoic acid at 10.6% compared to the tall oil fatty acids at 32.2% and the acetic acid 4.6%. The acetic acid, tall oil fatty acids and the 2-ethyhexanoic acid, have low acute oral toxicity compared to the n-tallow alkyltrimethyenediamine. Therefore the acute oral toxicity will come essentially from the main (52.2%) component in the reaction mass, the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine. Therefore the assessment for acute oral toxicity for the reaction mass can be based on this substance. There is data on this substance but from relatively old reports, there is also supporting data from the structural analogue N-(Hydrogenated tallow)-1,3-diaminopropane (CAS NO 68603-64-5). This based on hydrogenated tallow so it is also predominantly C18 and C18 saturated so less C18 unsaturated than N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine target substance biut still suitable as supportive data.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: HsdRccHan: WIST (SPF)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Weight at study initiation:
Step 1: 172-180 g
Step 2: 176-177 g

- Fasting period before study: overnight and 3-4 hours after dosing
- Housing: macrolon cages on Altromin saw fibre bedding
- Diet: ad libitum (Altromin 1324 (TPF)
- Water: ad libitum (drinking water)
- Acclimation period: at least 5 days
- Other: marked individually by tail painting, SPF-animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
Step 1: 1.0 g/ 5 mL
2.0 g/ 10 mL
Step 2: 2.0 g/ 10 mL

- Amount of vehicle: 10 mL/ kg body weight
- Justification for choice of vehicle: non-toxic and lipophil
- Lot/batch no. : Lot 067K0116, Sigma Chemicals

MAXIMUM DOSE VOLUME APPLIED: 1.80 mL

DOSAGE PREPARATION : freshly mixed to homogeneity prior to administration

CLASS METHOD
- Rationale for the selection of the starting dose:
the starting dose was choosen to be 2000 mg/kg bw

Doses:

Step 1: 2000 mg/kg bw
Step 2: 2000 mg/kg bw

No. of animals per sex per dose:

Step 1 (2000 mg/kg bw): 3 female rats
Step 2 (2000 mg/kg bw): 3 female rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: A careful clinical examination was made several times on the day of dosing.
Parts of this were at least three observations within the first four hours post-dose.
Animals were observed once a day thereafter.
The animals were weighed prior to the administration and once a week thereafter.

- Necropsy of survivors performed: yes

- Other examinations performed: Cage side observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity
and behaviour pattern were examined. Particular attention was directed to observations of tremor,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

not applicable

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

2 500 mg/kg bw

Remarks on result:

other: acute toxic class method; according to GHS (Globally Harmonized Classification System) the test item N-(Hydrogenated tallow)-1,3-diaminopropane was classified into Category 5

Mortality:

Step 1 (2000 mg/kg bw): 28 h post-dose one animal was found dead

Clinical signs:

other: see remarks on results including tables and figures

Gross pathology:

Animal no. 3 of step 1 (2000 mg/ kg bw):
The dead animal was found in a lateral position. Stomach: solid content.
Small and large intestine: partly filled with solid substance.
Animal no.2 of step 2 (2000 mg/ kg bw):
The spleen was enlarged, with a weight of 0.872 g.

Signs of toxicity related to dose level used, time of onset and duration:

Animal no. 1 of step 1 (2000 mg/kg bw): 20 min as well as 125 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 21 h 05 min as well as 27 h 5 min, 45 h 05 min post-dose: slightly reduced spontaneous activity, piloerection. 51 h 5 min post-dose: slightly reduced spontaneous activity. 69 h 05 min until the end of the observation period: no further symptoms were observed.

Animal no. 2 of step 1 (2000 mg/kg bw): 30 min as well as 3 h post-dose: slightly reduced spontaneous activity, apathy, piloerection. 24 h as well as 45 h post-dose: slightly reduced spontaneous activity, piloerection. 71 h as well as 94 h, 99 h post-dose: piloerection 117 h until the end of the observation period: no further symptoms were observed.

Animal No. 3 of step 1 (2000 mg/kg bw): 30 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 2 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, salivation. 4 h post-dose: moderately reduced spontaneous activity, apathy, piloerection, salivation. 28 h post-dose: this animal was found dead.

Animal no. 1 of step 2 (2000 mg/kg bw):1 h post-dose: slightly reduced spontaneous activity, apathy, piloerection, stertorousness. 3 h as well as 22 h, 27 h post-dose: piloerection. 45 h until the end of the observation period: no further symptoms were observed.

Animal no. 2 of step 2 (2000 mg/kg bw): 1 h post-dose: slightly reduced spontaneous activity, apathy, piloerection. 3 h as well as 22 h, 27 h, 45 h post-dose: piloerection. 69 h until the end of the observation period: no further symptoms were observed.

Animal no.3 of step 2 (2000 mg/kg bw): 1 h post-dose: piloerection. 4 h as well as 23 h, 28 h, 46 h post-dose: piloerection. 69 h until the end of the observation period: no further symptoms were observed.

Absolute body weights in g :

Animal No.	Sex	Day 0	Day 7	Day 14
Step 1 (2000 mg/kg bw)
1	female	180	195	220
2	female	173	154	193
3	female	172	157-this animal was found dead.
Step 2 (2000 mg/kg bw)
1	female	177	158	195
2	female	177	178	197
3	female	176	184	190

Interpretation of results:

harmful

Remarks:

Migrated information criteria given in Annex VI of the Directive 2001/59/EC Criteria used for interpretation of results: EU

Conclusions:

Considering the reported data of this toxicity test it can be stated that the test item N-(Hydrogenated tallow)-1,3-diaminopropane showed oral toxic
characteristics.
According to GHS (Globally Harmonized Classification System) the test item N-(Hydrogenated tallow)-1,3-diaminopropane was classified into Category 5.
(LD50 cutoff:300 mg/kg bw).

Executive summary:

The acute toxic class method for oral toxicity was performed on nulliparous female HsdRccHan:WIST rats, who received a single exposure of N-(Hydrogenated tallow)-1,3-diaminopropane via oral gavage.

A careful clinical examination was made several times on the day of dosing. Parts of this were at least three observations within the first four hours post-dose. Animals were observed once a day thereafter for 14 days. Cage side observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross necropsy. All gross pathological changes were recorded.

In step 1 three female rat were dosed by oral gavage with 2000 mg N-(Hydrogenated tallow)-1,3-diaminopropane/ kg body weight in cotton seed oil. One of the animals died 28 h post-dose. Signs of slight toxicity indicated by apathy, piloerection and reduced spontaneous activity were observed during the first 3 and 5 days in animal 1 and 2 of step 1, respectively. No further symptoms were observed in these two animals until the end of the observation period. In animal 3 apathy, piloerection, reduced spontaneous activity and salvation were evident after dosing and the animal was found dead 28 h post-dose. In step 2 three additional female rats were dosed with 2000 mg N-(Hydrogenated tallow)-1,3-diaminopropane/ kg body weight. No compound related mortality was recorded for any of these animals. Observed signs of toxicity were reduced spontaneous activity, apathy, pilerection and stertorousness. All animals of step 2 recovered within 3 days. According to the toxic class regime no further testing was required.

Following pathological changes were observed in animal 3 of step 1 (2000 mg/kg bw): The dead animal was found in a lateral position. The stomach as well as small and large intestine were at least partly filled with solid content. Following pathological changes were observed in animal 2 of step 2 (2000 mg/kg bw): The spleen was enlarged, with a weight of 0.872 g.

Considering the reported data of this toxicity test it can be stated that the test item N-(Hydrogenated tallow)-1,3-diaminopropane showed oral toxic characteristics. On basis of the test results and in conformity with the criteria given in Annex VI of the Directive 2001/59/EC the substance should be classified as harmful. According to GHS (Globally Harmonized Classification System) the test item N-(Hydrogenated tallow)-1,3-diaminopropane was classified into Category 5 (LD₅₀cut-off: 2500 mg/kg bw).

The acute oral toxicity test was conducted under GLP according to the guidelines for the acute toxic class method OECD 423, EU method B1.tris and OPPTS 870.110 without deviation.

Reference 18

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2007-11-27 to 2008-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The starting material for this reaction mass is n-tallow alkyltrimethyenediamine CAS No 61791-55-7, which was registered for REACH as N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine(CAS No 1219010-04-4), that is reacted with tall oil fatty acids (CAS No 61790-12-3), 2-ethylhexanoic acid (CAS No 149-57-5) and acetic acid (CAS No 64-18-7). The n-tallow alkyltrimethyenediamine is ca. 52.2% of the reaction mixture with the 2-ethylhexanoic acid at 10.6% compared to the tall oil fatty acids at 32.2% and the acetic acid 4.6%. The acetic acid, tall oil fatty acids and the 2-ethyhexanoic acid, have low acute oral toxicity compared to the the n-tallow alkyltrimethyenediamine. Therefore the acute oral toxicity will come essentially from the main (52.2%) component in the reaction mass, the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine. Therefore the assessment for acute oral toxicity for the reaction mass can be based on this substance. There is data on this substance but from relatively old reports, there is a supporting study for the close structural analogue (Z)-N-9-Octadecenylpropane-1,3-diamine CAS No 7173-62-8 paste. This is also C16 and C18 but it is predominantly C18 and the C18 is almost all unsaturated rather than the lower level in the target molecule. The higher degree of unsaturation in the source substance increases its reactivity and therefore its acute oral toxicity, it is therefore a worst case when used as a source for read across to the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine target substance.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: HsdRccHan:WIST (Full-Barrier)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Weight at study initiation:
Step 1: 176-194 g
Step 2: 172-184 g
Step 3: 158-176 g

- Fasting period before study: overnight
- Housing: Macrolon cages on Altromin saw fiber bedding
- Diet: ad libitum, Altromin 1324 (TPF)
- Water: ad libitum (drinking water)
- Acclimation period: at least 5 days
- other: SPF animals, marked individually by tail painting

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
Step 1: 1 g/ 5 mL and 2 g/10 mL cotton seed oil
Step 2: 0.3 g/ 10 mL cotton seed oil
Step 3: 0.3 g/ 10 mL cotton seed oil
- Amount of vehicle: 10 mL/ kg body weight
- Justification for choice of vehicle: non-toxic characteristic
- Lot/batch no.: Lot 067K0116, sigma Chemicals

MAXIMUM DOSE VOLUME APPLIED: 1.94 mL

CLASS METHOD
- Rationale for the selection of the starting dose: 2000 mg/ kg was selected as the starting dose

Doses:

Step 1: 2000 mg/ kg
Step 2 and 3: 300 mg/ kg body weight

No. of animals per sex per dose:

female nulliparous rats
2000 mg/ kg dose (Step 1): 3 animals
300 mg/ kg dose (Step 2 and 3): 3 animals + 3 animals

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: A careful clinical examination was made several times on the day of dosing.
Parts of this were at least three observations within the first four hours post-dose.
Animals were observed once a day thereafter.
The animals were weighed prior to the administration and once a week thereafter.

- Necropsy of survivors performed: yes

- Other examinations performed: Cage side observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity
and behaviour pattern were examined. Particular attention was directed to observations of tremor,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

not applicable

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

1 000 mg/kg bw

Remarks on result:

other: acute toxic class method; according to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4

Mortality:

Step 1 (2000 mg/kg bw): 2 animals were found dead, one after 17.5 h and one after 76.25 h
Step 2 (300 mg/kg bw): no mortality observed
Step 3 (300 mg/kg bw): no mortality observed

Clinical signs:

other: see remarks on results including tables and figures

Gross pathology:

Animal no. 1 of step 1 (2000 mg/ kg bw):
The dead animal was found in a lateral position. Small and large intestine had
liquid content.
Animal no. 2 of step 1 (2000 mg/ kg bw):
The animal was found in a face down position. The anus was full of faeces.
The stomach contained rests of the test item and was bloated.

Signs of toxicity related to dose level used, time of onset and duration:

Animal no. 1 of step 1(2000 mg/kg bw): 05 min post-dose: salivation. 3 h 45 min post-dose: apathy, piloerection. 20 h 45 min as well as 26 h 15 min post-dose: slightly reduced spontaneous activity, apathy, piloerection. 44 h 45 min post-dose: slightly reduced spontaneous activity, apathy, piloerection, diarrhea. 48 h 45 min post-dose: slightly reduced spontaneous activity, apathy, piloerection, diarrhea, weight loss. 76 h 15 min post-dose: the animal was found dead.

Animal no. 2 of step 1(2000 mg/kg bw): 30 min post-dose: slightly reduced spontaneous activity, piloerection. 17 h 30 min post-dose: the animal was found dead.  

Animal no. 3 of step 1 (2000 mg/kg bw): 30 min post-dose: slightly reduced spontaneous activity, piloerection, salivation. 17 h 30 min as well as 41 h 15 min, 48 h 30 min post-dose: moderately reduced spontaneous activity, piloerection, diarrhea, stertorousness. 66 h post-dose: moderately reduced spontaneous activity, piloerection, diarrhea. 93 h 30 min as well as 114 h, 138 h post-dose: slightly reduced spontaneous activity, piloerection, respiratory sounds, diarrhea. 166 h as well as 191 h post-dose: slightly reduced spontaneous activity, piloerection 217 h as well as 238 h 15 min 257 h post-dose: piloerection. 12 days post-dose until the end of the observation period: no further symptoms were observed.  

Animal No. 1 of step 2 (300 mg/kg bw 45 min as well as 21 h 45 min post-dose: slightly reduced spontaneous activity, piloerection. 2 h 45 min post-dose: piloerection, half-eyelid closure. 26 h 45 min as well as 44 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, bloody snout. 68 h 45 min as well as 92 h 45 min post-dose: piloerection. 119 h 45 min post-dose: piloerection, chromodakryorrhoe. 145 h 15 min post-dose: piloerection, chromodakryorrhoe, heavy respiration, articulation, convulsion. 164 h 45 min as well as 189 h 45 min, 213 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, stertorousness, weight loss. 236 h 45 min as well as 260 h 45 min, 284 h 45 min post-dose: slightly reduced spontaneous activity, piloerection, stertorousness. 13 days until the end of the observation period: no further symptoms were observed.

Animal no. 2 of step 2 (300 mg/kg bw): 45 min as well as 2 h 45 min, 21 h 45 min, 26 h 15 min, 44 h 45 min postdose: slightly reduced spontaneous activity, piloerection. 68 h 45 min as well as 92 h 45 min post-dose: piloerection. 5 days until the end of the observation period: no further symptoms were observed.

Animal no.3 of step 2 (300 mg/kg bw): 45 min as well as 2 h 45, 21 h 45, 26 h 45 min, 44 h 45 post-dose: slightly reduced spontaneous activity, piloerection. 68 h 45 min as well as 92 h 45 min post-dose: piloerection. 119 h 45 min post-dose: piloerection, chromodakryorrhoe. 145 h 15 min post-dose: piloerection, chromodakryorrhoe, heavy respiration, articulation, convulsion. 7 days post-dose until the end of the observation period: no further symptoms were observed.

Animal no. 1,2 and 3 of step 3 (300 mg/kg bw 30 min as well as 2 h 30 min post-dose: slightly reduced spontaneous activity, piloerection. 19 h 30 min as well as 43 h 30 min, 67 h 30 min, 94 h 30 min post-dose: piloerection. 5 days post-dose until the end of the observation period: no further symptoms were observed.  

Absolute body weights in g

Animal No.	Sex	Day 0	Day 7	Day 14
Step 1 (2000 mg/kg bw)
1	female	177	149- This animal was found dead.
2	female	194	185- This animal was found dead.	.
3	female	176	138	178
Step 2 (300 mg/kg bw)
1	female	184	145	150
2	female	172	193	200
3	female	172	162	188
Step 3 (300 mg/kg bw)
1	female	173	190	203
2	female	158	179	187
3	female	176	198	211

Interpretation of results:

harmful

Remarks:

Migrated information criteria given in Annex VI of the Directive 2001/59/EC Criteria used for interpretation of results: EU

Conclusions:

Considering the reported data of this toxicity test it can be stated that the test item N-Oleyl-1,3-diaminopropane showed acute oral toxic characteristics. According to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4 (LD50
cut-off: 1000 mg/kg bw).

Executive summary:

The acute toxic class method for oral toxicity was performed on nulliparous female HsdRccHan:WIST rats, who received a single exposure of N-Oleyl-1,3-diaminopropane (liquid/paste) via oral gavage.

A careful clinical examination was made several times on the day of dosing. Parts of this were at least three observations within the first four hours post-dose. Animals were observed once a day thereafter for 14 days. Cage side observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross necropsy. All gross pathological changes were recorded.

In step 1 three female rats were dosed by oral gavage with 2000 mg N-Oleyl-1,3-diaminopropane/ kg body weight in cotton seed oil. Severe toxicity indicated by salivation, apathy, piloerection, reduced spontaneous activity, diarrhoea and weight loss was observed in this group after the application. Animal 1 and 2 died within 17.5 h and 76.25 h, respectively. Animal 3 recovered on day 12 and survived the 14 days observation period. In step 2 three female rats were dosed with 300 mg N-Oleyl-1,3-diaminopropane/ kg body weight. Animal 1 of step 2 showed signs of toxicity indicated by reduced spontaneous activity, half eyelid-closure, piloerection, bloody snout, chromodakryorrhoe, heavy respiration, articulation, convulsion, weight loss and stertorousness and recovered on day 13. Animal 2 of step 2 displayed reduced spontaneous activity and piloerection and recovered on day 5. Animal 3 of step 2 showed following signs of toxicity: piloerection, reduced spontaneous activity, chromodakryorrhoe, heavy respiration, articulation and convulsion. No further symptoms were observed from day 7 on. In step 3 three additional female rats were dosed with 300 mg N-Oleyl-1,3 -diaminopropane/ kg body weight. All three animals showed signs of toxicity indicated by reduced spontaneous activity and piloerection. No more symptoms were observed from day 5 until the end of the observation period. According to the toxic class regime no further testing was required.

Following pathological changes were observed. Animal 1 of step 1 (2000 mg/kg bw) was found dead in a lateral position after 3 days and its small and large intestine had liquid content. Animal 2 of step 1 (2000 mg/kg bw) was found dead in a face down position after 17.5 h. Its anus was full of faeces and the stomach contained rests of the test item and was bloated.

Considering the reported data of this toxicity test it can be stated that the test item N-Oleyl-1,3-diaminopropane showed acute oral toxic characteristics. On basis of the test results and in conformity with the criteria given in Annex VI of the Directive 2001/59/EC the substance should be classified as harmful. According to GHS (Globally Harmonized Classification System) the test item N-Oleyl-1,3-diaminopropane was classified into Category 4 (LD₅₀ cut-off: 1000 mg/kg bw).

The acute oral toxicity test was conducted under GLP according to the guidelines for the acute toxic class method OECD 423, EU method B1.tris and OPPTS 870.110 without deviation.

Reference 19

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2007-11-27 to 2008-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Justification for type of information:

The starting material for this reaction mass is n-tallow alkyltrimethyenediamine CAS No 61791-55-7, which was registered for REACH as N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine(CAS No 1219010-04-4), that is reacted with tall oil fatty acids (CAS No 61790-12-3), 2-ethylhexanoic acid (CAS No 149-57-5) and acetic acid (CAS No 64-18-7). The n-tallow alkyltrimethyenediamine is ca. 52.2% of the reaction mixture with the 2-ethylhexanoic acid at 10.6% compared to the tall oil fatty acids at 32.2% and the acetic acid 4.6%. The acetic acid, tall oil fatty acids and the 2-ethyhexanoic acid, have low acute oral toxicity compared to the the n-tallow alkyltrimethyenediamine. Therefore the acute oral toxicity will come essentially from the main (52.2%) component in the reaction mass, the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine. Therefore the assessment for acute oral toxicity for the reaction mass can be based on this substance. There is data on this substance but from relatively old reports, there is also supporting data from the structural analogue Amines, N-C12-14-alkyltrimethylenedi- (CAS NO 90640-43-0). The shorter carbon chain length makes this a worst case for read across to the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine target substance.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: HsdRccHan: WIST (SPF)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Weight at study initiation:
Step 1: 168 g (1 animal)
Step 2: 189-200 g
Step 3: 133-173 g
Step 4: 180-214 g

- Fasting period before study: overnight and 3-4 hours after dosing
- Housing: macrolon cages on Altromin saw fibre bedding
- Diet: ad libitum (Altromin 1324 (TPF)
- Water: ad libitum (drinking water)
- Acclimation period: at least 5 days
- Other: marked individually by tail painting, SPF animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
Step 1: 1.0 g/ 5 mL
Step 2: 0.3 g/ 10 mL
Step 3: 0.05 g/ 10 mL
Step 4: 0.05 g/ 10 mL

- Amount of vehicle: 10 mL/ kg body weight
- Justification for choice of vehicle: non-toxic and lipophil
- Lot/batch no. : Lot 067K0116, Sigma Chemicals

MAXIMUM DOSE VOLUME APPLIED: 2.00 mL

DOSAGE PREPARATION : freshly mixed to homogeneity prior to administration

CLASS METHOD
- Rationale for the selection of the starting dose:
preliminary test with one animal performed (2000 mg/kg), due to immeadiate preacute lethality
the starting dose was choosen to be 300 mg/kg bw

Doses:

Step 1: 2000 mg/kg bw
Step 2: 300 mg/kg bw
Step 3: 50 mg/kg bw
Step 4: 50 mg/kg bw

No. of animals per sex per dose:

Step 1 (2000 mg/kg bw): 1 female rat
Step 2 (300 mg/kg bw): 3 female rats
Step 3 (50 mg/kg bw): 3 female rats
Step 4 (50 mg/kg bw): 3 female rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: A careful clinical examination was made several times on the day of dosing.
Parts of this were at least three observations within the first four hours post-dose.
Animals were observed once a day thereafter.
The animals were weighed prior to the administration and once a week thereafter.

- Necropsy of survivors performed: yes

- Other examinations performed: Cage side observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity
and behaviour pattern were examined. Particular attention was directed to observations of tremor,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

not applicable

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

200 mg/kg bw

Remarks on result:

other: acute toxic class method; according to GHS (Globally Harmonized Classification System) the test item N-C12,14 alkyl-1,3-diaminopropane was classified into Category 3

Mortality:

Step 1 (2000 mg/kg bw, 1 female rat): within 1 h 30 min post-dose the animal was found dead
Step 2 (300 mg/kg bw): within 2 days post-dose all three animals were found dead
Step 3 (50 mg/kg bw): no deaths observed within the observation period
Step 4 (50 mg/kg bw): 1 animal was found dead 13 days post-dose

Clinical signs:

other: see remarks on results including tables and figures

Gross pathology:

Animal no. 1 of step 1 (2000 mg/ kg bw):
The stomach, small and large intestine were bloody.
Animal no.1 of step 2 (300 mg/ kg bw):
The stomach was bloody. The stomach and small intestines had a yellow content. The liver had a
dark colour.
Animal no.2 of step 2 (300 mg/ kg bw):
The animal was found in a face down position. The stomach mucosa was bloody and the stomach
content was yellow. The anus was smeared with faeces.
Animal no.3 of step 2 (300 mg/ kg bw):
The animal was found in a face down position. The stomach mucosa was
bloody and the stomach content was yellow.
Animal no. 2 of step 4 (50 mg/ kg bw):
Small and large intestine and the stomach were bloated. The spleen weighed 150 g.

Signs of toxicity related to dose level used, time of onset and duration:

Animal no. 1 of step 1(2000 mg/kg bw): 05 min post-dose: apathy, salivation. 20 min post-dose: moderately reduced spontaneous activity, salivation, diarrhoea, face down position. 1 h 30 min post-dose: this animal was found dead.

Animal no. 1 of step 2(300 mg/kg bw): 1 h 30 min post-dose: slightly reduced spontaneous activity, piloerection. 3 h 30 min post-dose: moderately reduced spontaneous activity, piloerection, diarrhoea. 22 h 30 min post-dose: moderately reduced spontaneous activity, piloerection, diarrhoea, sunken flank. 26 h 30 min: this animal was found dead.

Animal no. 2 of step 2 (300 mg/kg bw): 30 min as well as 3 h post-dose: slightly reduced spontaneous activity, apathy, piloerection. 24 h as well as 45 h post-dose: slightly reduced spontaneous activity, piloerection. 71 h as well as 94 h, 99 h post-dose: piloerection 117 h until the end of the observation period: no further symptoms were observed.

Animal No. 3 of step 2 (300 mg/kg bw): 45 min post-dose: slightly reduced spontaneous activity, piloerection, diarrhoea. 17 h 45 min post-dose: moderately reduced spontaneous activity, piloerection, diarrhoea. 41 h 15 min post-dose: this animal was found dead.

Animal no. 1 of step 3 (50 mg/kg bw): 45 min post-dose: slightly reduced spontaneous activity, piloerection. 17 h 45 min post-dose: moderately reduced spontaneous activity, piloerection, diarrhoea. 41 h 15 min post-dose: this animal was found dead.

Animal no. 2 of step 3 (50 mg/kg bw): 1 h, 20 h, 44 h post-dose: slightly reduced spontaneous activity, piloerection. 48 h post-dose: moderately reduced spontaneous activity, piloerection. 68 h, 92 h, 116 h post-dose: slightly reduced spontaneous activity, piloerection. 6 days post-dose until the end of the observation period: no further symptoms were observed.

Animal no.3 of step 3 (50 mg/kg bw): 2 h 30 min as well as 21 h 30 min, 46 h 30 min post-dose: piloerection. 4 h post-dose: slightly reduced spontaneous activity, piloerection. 3 days post-dose until the end of the observation period: no further symptoms were observed.

Animal no. 1 of step 4 (50 mg/kg bw): 3 h post-dose: slightly reduced spontaneous activity, piloerection. 21 h post-dose: slightly reduced spontaneous activity, piloerection, diarrhea. 45 h 30 min post-dose: slightly reduced spontaneous activity, piloerection. 69 h 30 min post-dose: piloerection. 97 h post-dose: slight piloerection, respiratory sounds. 119 h post-dose: piloerection. 6 days until the end of the observation period: no further symptoms were observed.

Animal no. 2 of step 4 (50 mg/kg bw): 3 h post-dose: slightly reduced spontaneous activity, piloerection. 21 h post-dose: slightly reduced spontaneous activity, piloerection, diarrhea. 45 h 30 min post-dose: slightly reduced spontaneous activity, piloerection. 69 h 30 min post-dose: piloerection. 93 h 30 min as well as 119 h post-dose: piloerection. 142 h 30 min post-dose: no symptoms observed. 11 days post-dose: slightly reduced spontaneous activity, piloerection. 12 days post-dose: slightly reduced spontaneous activity, piloerection, respiratory sounds. 13 days post-dose: this animal was found dead.

Animal no.3 of step 4 (50 mg/kg bw): 3 h as well as 69 h 30 min, 93 h 30 min, 119 h post-dose: piloerection. 3 h 30 min as well as 45 h 30 min: slightly reduced spontaneous activity, piloerection. 21 h post-dose: slightly reduced spontaneous activity, piloerection, diarrhea. 142 h 30 min post-dose until the end of the observation period: no further symptoms were observed.

Absolute body weights in g :

Animal No.	Sex	Day 0	Day 7	Day 14
Step 1 (2000 mg/kg bw)
1	female	168	This animal was found dead.
Step 2 (300 mg/kg bw)
1	female	189	This animal was found dead.
2	female	200	188- This animal was found dead.	.
3	female	199	192- This animal was found dead.
Step 3 (50 mg/kg bw)
1	female	173	205	206
2	female	133	162	177
3	female	146	163	174
Step 4 (50 mg/kg bw)
1	female	206	200	196
2	female	180	182	This animal was found dead.
3	female	217	252	255

Interpretation of results:

toxic

Remarks:

Migrated information criteria given in Annex VI of the Directive 2001/59/EC Criteria used for interpretation of results: EU

Conclusions:

Considering the reported data of this toxicity test it can be stated that the test item N-C12,14 alkyl-1,3-diaminopropane showed acute oral toxic
characteristics.
According to GHS (Globally Harmonized Classification System) the test item N-C12,14 alkyl-1,3-diaminopropane was classified into Category 3
(LD50 cut-off: 200 mg/kg bw).

Executive summary:

The acute toxic class method for oral toxicity was performed on nulliparous female HsdRccHan:WIST rats, who received a single exposure of N-C12,14 alkyl-1,3-diaminopropane via oral gavage.

A careful clinical examination was made several times on the day of dosing. Parts of this were at least three observations within the first four hours post-dose. Animals were observed once a day thereafter for 14 days. Cage side observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. At the end of the observation period the animals were sacrificed by an overdosage of pentobarbital. All animals were subjected to gross necropsy. All gross pathological changes were recorded.

In step 1 single female rat was dosed by oral gavage with 2000 mg N-C12,14 alkyl-1,3-diaminopropane/ kg body weight in cotton seed oil. Severe, immediate toxicity indicated by salivation, face down position, diarrhoea and moderately reduced spontaneous activity was observed in this animal, followed by death at 1 h and 30 min post-dose. In step 2 three female rats were dosed with 300 mg N-C12,14 alkyl-1,3-diaminopropane/ kg body weight. All three animals showed severe signs of toxicity indicated by piloerection, sunken flank, diarrhoea and reduced spontaneous activity and died within 2 days post-dose. In step 3 three female rats were dosed with 50 mg N-C12,14 alkyl-1,3-diaminopropane/ kg body weight. No mortality was observed in all three animals.

Observed signs of toxicity were reduced spontaneous activity, piloerection and diarrhoea. The animals had recovered within 3 (animal 2) to 6 days (animal 1 and 3) post-dose and showed no symptoms thereafter. In step 4 three additional female rats were dosed with 50 mg N-C12,14 alkyl-1,3-diaminopropane/ kg body weight. Mortality was observed in one animal. Observed signs of toxicity were reduced spontaneous activity, piloerection and diarrhoea. All three animals recovered within 6 days post-dose. Animal 1 and 3 of step 4 showed no further symptoms thereafter. Animal 2 of step 4 showed the same toxicity signs again from day 11 on and was found dead on day 13. According to the toxic class regime no further testing was required.

Following pathological changes were observed in the step 1 animal (2000 mg/kg bw): The stomach, small and large intestine were bloody. Animal 1 of step 2 (300 mg/kg bw) exhibited a bloody stomach. The stomach and small intestines had a yellow content. The liver had a dark colour. Animal 2 of step 2 (300 mg/kg bw) was found in a face down position and exhibited a bloody stomach mucosa and the stomach content was yellow. The anus was smeared with faeces. Animal 3 of step 2 (300 mg/kg bw) was found in a face down position and exhibited a bloody stomach mucosa and a yellow stomach content. In animal 2 of step 4 (50 mg/kg bw) the small and large intestine and the stomach were bloated. The spleen weight was markedly increased (150 g).

Considering the reported data of this toxicity test it can be stated that the test item N-C12,14 alkyl-1,3-diaminopropane showed acute oral toxic characteristics. On basis of the test results and in conformity with the criteria given in Annex VI of the Directive 2001/59/EC the substance should be classified as toxic. According to GHS (Globally Harmonized Classification System) the test item N-C12,14 alkyl-1,3-diaminopropane was classified into Category 3 (LD₅₀ cut-off: 200 mg/kg bw).

The acute oral toxicity test was conducted under GLP according to the guidelines for the acute toxic class method OECD 423, EU method B1.tris and OPPTS 870.110 without deviation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

500 mg/kg bw

Quality of whole database:

High. Consistent and reliable data over category.

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The starting material for this reaction mass is n-tallow alkyltrimethyenediamine CAS No 61791-55-7, which was registered for REACH as N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine(CAS No 1219010-04-4), that is reacted with tall oil fatty acids (CAS No 61790-12-3), 2-ethylhexanoic acid (CAS No 149-57-5) and acetic acid (CAS No 64-18-7). The n-tallow alkyltrimethyenediamine is ca. 52.2% of the reaction mixture with the 2-ethylhexanoic acid at 10.6% compared to the tall oil fatty acids at 32.2% and the acetic acid 4.6%.  The acetic acid, tall oil fatty acids and the 2-ethyhexanoic acid, have low acute oral toxicity compared to the n-tallow alkyltrimethyenediamine. Therefore the acute oral toxicity will come essentially from the main (52.2%) component in the reaction mass, the N-C16-18-alkyl(evennumbered) C18 unsaturated propane-1, 3-diamine. Therefore the assessment for acute oral toxicity for the reaction mass can be based on this substance. 

Oral

A total of 14 studies were available for evaluation of the acute oral toxicity of Diamine C16-18, C18-unsaturated. Of these, 9 studies evaluated the acute toxicity of Diamine C16-18, C18-unsaturated itself. One of these, on Duomeen T (Kynoch 1984)is considered to be supportive, and resulted to an LD50 in rats of 945 mg/kg bw. The other studies generally showed much lower toxicity, but the test substance is not clearly described and could possibly be formulations, these studies are not further considered.   Data more relevant for the classification is derived from series of highly reliable GLP studies performed together over the category of alkyl-diamines. (Also see ‘Justification in support of cross-reading between Diamines’, document Support Cross-reading - Diamines Final 20131220.pdf attached to IUCLID chapter 13)   Alkyl chain Study Physical state Oral LD₅₀cut-off mg/kg bw Oleyl RA from 7173-62-8, Acute toxicity: oral_liquid, Haist 2008 liquid 500 Oleyl RA from 7173-62-8 Acute toxicity: oral_paste, Haist 2008 liquid/ paste 1000 C_12-14 RA from 90640-43-0, Acute toxicity: oral. Haist 2008 liquid/ paste 200 HT RA from 68603-64-5, Acute toxicity: oral. Haist, 2008 solid 2500 Coco RA from 61791-63-7, Acute toxicity: oral. Haist 2008 liquid 300 Tallow read across to Oleyl paste 500-1000   For acute oral toxicity, a relation can be observed with chain length and level of unsaturation: acute toxicity become less with increasing lengths of alkyl chains, and related with increasing levels of unsaturation. The diamines based on relative short chains up to C₁₄, Cat.3 classification follows. The diamines based on higher alkyl chain lengths are classified as Cat.4 when also a high level of unsaturation is present. In HT-diamine with fully saturated longer alkyl chains of C_16-18, has a LD50 cut-off 2500, and falls in GHS Cat.5, but no classification is needed for CLP.   Diamine C16-18, C18-unsaturated can in this case best be compared to Oleyl diamine. Although the chain-length distribution is the same as for tallow, the presence of some unsaturated C18, makes cross-reading to Oleyl more appropriate.   Inhalation: There is no study on inhalation toxicity available for N-C16-18-alkyl-(evennumbered) C18 unsaturated) propane-1,3-diamine. REACH stipulates that testing by the inhalation route is appropriate if exposure of humans via inhalation is likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size. REACH guidance R.7.a, chapter. 7.4 Acute toxicity, indicates that in principle no inhalation studies are needed when vp < 0.1 Pa at 20°C or particle size > 100 µm.Diamine C16-18, C18-unsaturatedis a paste with a vapour pressure less than 0.0015 Pa at 20°C (value based on read-across from shorter chain C_12-14-diamine). Also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur, and no acute inhalation test was performed.   There is no risk for aspiration as with a mp around 37 °C, Diamine C16-18, C18-unsaturatedis a paste upon ingestion.   Dermal: There is no dermal LD 50 value for acute skin toxicity of N-C16-18-alkyl-(evennumbered) C18 unsaturated) propane-1,3-diamine, and due to the corrosive properties of the substance it is not ethical to carry out this animal study. A study evaluating the acute dermal toxicity N-Coco-1,3-diaminopropane according has shown mortality in 1 out of 10 rabbits at a limit dose of 2000 mg/kg bw. Low systemic toxicity via dermal route can therefore also be expected forDiamine C16-18, C18-unsaturated. Use and handling of the pure substance is only industrial and professional (formulation), and the classification as irritant of the substance requires risk management methods which eliminate the risk of acute systemic toxicity from potential for skin contact.

Justification for classification or non-classification

An LD50 value of 500-1000 mg/kg will lead to CLP Category 4 for acute oral toxicity.

Dermal systemic toxicity is expected to be low, while severe irritating properties will limit likelihood of exposures.

Related to low vp and no inhalable particles, there will be low likelihood of exposures via inhalation.

No classification STOT-SE Cat.3 needed:

The active substance is not structurally related to any known class of neurotoxic chemicals. In addition, repeated dose studies did not show indications of specific neurotoxicity, in specific neurotoxicity measures as sensory activity, grip strength, and motor activity assessment.

Diamine C16-18, C18-unsaturatedis a paste with mp of 37°C and a vapour pressure less than 0.0015 Pa at 20°C (value based on read-across from shorter chain C_12-14-diamine). Also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur.