N1,N3-diallylpropane-1,3-diamine dihydrochloride

Endpoint summary

Description of key information

In an acute oral toxicity study conducted according to OECD TG 423, the LD50 cut-off in rats was determined to be 500 mg/kg bw. In addition, in an acute dermal toxicity study conducted according to OECD TG 402, the LD50 for acute dermal toxicity in rats was > 2000 mg/kg bw. Testing for acute inhalation toxicity was not performed because results from two routes of exposure are already provided and relevant exposure via the inhalation route is not very likely due to particle size and vapour pressure of the substance.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-04-16 to 2020-08-13

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)

Version / remarks:

adopted 17th December 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- In order to get the test item in a solution or suspension, which is applicable to the animals, aqua ad injectionem (Deltamedica, lot no. 901110, expiry date: December 2021) was evaluated as vehicle and was considered to be adequate. This vehicle was chosen due to its non-toxic characteristics.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution/suspension

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-10 weeks
- Weight at study initiation: Step 1 (300 mg/kg bw): 164–168 g; Step 2 (2000 mg/kg bw): 177–191 g; Step 3 (300 mg/kg bw): 119–149 g
- Fasting period before study: Prior to the administration of the test material, food was withheld from the test animals for 16 to 19 hours (access to water was permitted). Following the period of fasting the animals were weighed and the test item was administered. Food was provided again approximately 4 hours post dosing.
- Housing: The animals were kept in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water (sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: at least five days under laboratory conditions
- Microbiological status when known : The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.

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:

water

Details on oral exposure:

VEHICLE
- Justification for choice of vehicle: the vehicle was chosen due to its non-toxic characteristics
- Lot/batch no. (if required): Deltamedica, lot no. 901110, expiry date: December 2021

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

Doses:

300 and 2000 mg/kg bw

No. of animals per sex per dose:

3 females per step

Control animals:

no

Details on study design:

- Duration of observation period following administration: The surviving animals were observed for 14 days after dosing for general clinical signs, morbidity and mortality. A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded. Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded. Cageside 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.
- Frequency of observations: A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). Thereafter, the animals were observed for clinical signs once daily until the end of the observation period.
- Frequency of weighing: The animals were weighed on day 1 (prior to the administration) and on days 8 and 15.
- Necropsy of survivors performed: The animal which had to be sacrificed for ethical reasons (animal no. 5, step 2) during the observation period was necropsied as soon as it was killed. At the end of the observation period the surviving animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally at a dosage of 250-400 mg/kg bw. All animals were subjected to gross necropsy and examined macroscopically for gross pathological changes. Macroscopic findings of the animal which had to be sacrificed for ethical reasons (animal no. 5, step 2) were red discoloured, fluid filled lungs. Due to these alterations lungs of animals no. 5 were preserved for a possible histopathological evaluation.

Statistics:

n.a.

Preliminary study:

n.a.

Key result

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

500 mg/kg bw

Based on:

test mat.

Mortality:

All animals treated with the test item at a dose of 300 mg/kg bw survived until the end of the study. Two animals treated with the test item at a dose of 2000 mg/kg bw died spontaneously on day 1 (animal no. 6) and on day 3 (animal no. 4) after treatment. One animal (no. 5) treated with the same dose level had to be sacrificed for ethical reasons on day 3 after treatment.

Clinical signs:

other: The six animals treated with the test item at a dose of 300 mg/kg bw showed no clinical findings of toxicological relevance. However, all animals of step 3 (animal no. 7, 8 and 9) showed a slight piloerection 1-2 hours after test item administration. All

Gross pathology:

At necropsy, no treatment-related macroscopic findings were observed in any surviving animals. Necropsy of the animal which had to be sacrificed for ethical reasons revealed diffuse red discoloured, fluid filled lungs (animal no. 5, step 2). Before the sacrifice the animal showed nasal discharge, which could be the initial reason for the reactions in the lungs by aspirating liquids into the respiratory tract. No other treated animal showed findings in the lungs. Due to these alterations lungs of animal no. 5 were preserved for a possible histopathological evaluation.

Other findings:

For details on results see Tables 1, 2 and 3 in box "Any other information on results incl. tables".

Table 1: Clinical signs

Step	Animal No./Sex	Starting dose (mg/kg bw)	Time of observation	Observations
1	1 / female	300	d 1 – d 15	nsf
	2 / female		d 1 – d 15	nsf
	3 / female		d 1 – d15	nsf
2	4 / female	2000	0 min – 30 min	nsf
			60 min	hunched posture, half eyelid closure, slightly reduced spontaneous activity
			120 min – 240 min	apathy, hunched posture, piloerection, half eyelid closure, slightly reduced spontaneous activity
			d 2	piloerection, half eyelid closure, slightly reduced spontaneous activity
			d 3	found dead
	5 / female		0 min – 30 min	nsf
			60 min – 180 min	apathy, hunched posture, slightly piloerection, half eyelid closure, slightly reduced spontaneous activity
			240 min	apathy, hunched posture, slightly piloerection, slightly reduced spontaneous activity
			d 2	slightly piloerection, half eyelid closure, slightly reduced spontaneous activity
			d 3	apathy, hunched posture, slightly piloerection, half eyelid closure, slightly reduced spontaneous activity, hyperesthesia, nasal discharge, euthanised for animal welfare reasons
	6 / female		0 min	nsf
			30 min	apathy, slightly reduced spontaneous activity,
			60 – 240 min	apathy, hunched posture, slightly piloerection, half eyelid closure, slightly reduced spontaneous activity
			360 min	found dead
3	7, 8, 9 / female	300	0 – 30 min	nsf
			60 – 120 min	slightly piloerection
			180 min – d 15	nsf

d = study day (study day 1 = day of administration); min = minute(s) post-application; nsf = no specific findings

Table 2: Individual data on necropsy findings

Step	Animal No. / Sex	Starting Dose (mg/kg bw)	Organ	Macroscopic Findings
1	1 / Female	300	-	nsf
	2 / Female		-	nsf
	3 / Female		-	nsf
2	4 / Female	2000	all examined organs	autolytic
	5 / Female		lung	diffuse red discoloured, fluid filled
	6 / Female		gastrointestinal tract	autolytic (fluid filled)
3	7 / Female	300	-	nsf
	8 / Female		-	nsf
	9 / Female		-	nsf

nsf = no specific findings

Table 3: Absolute Body Weights in g and Body Weight Change in %

	Step	Animal No. / Sex	Starting Dose (mg/kg bw)		BW (g)					Body Weight Change in Comparison to Day 1 (%)
					Day 1		Day 8		Day 15	Day 15
	1	1 / Female	300		168		194		206	23
		2 / Female			167		188		200	17
		3 / Female			164		189		199	21
	2	4 / Female	2000		191		n.a.*		n.a.*	n.a.*
		5 / Female			186		n.a.**		n.a.**	n.a.**
		6 / Female			177		n.a.***		n.a.***	n.a.***
	3	7 / Female	300		119		148		163	37
		8 / Female			145		178		193	33
		9 / Female			149		174		197	32

* found dead on day 3

** euthanised for animal welfare reasons on day 3

*** found dead on day 1

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

In an acute oral toxicity study in rats conducted according to OECD 423, the test material N1,N3-diallylpropane-1,3-diamine dihydrochloride did not show mortality or any signs of toxicity when administered at doses of 300 mg/kg bw. After treatment with 2000 mg/kg bw, two animals died spontaneously on day 1 and day 3 after treatment and one animal had to be sacrificed for ethical reasons. Based on the results, the LD50 cut-off value is therefore considered to be 500 mg/kg bw.

Executive summary:

In an acute oral toxicity study conducted according to OECD 423 (acute toxic class method), groups of female Wistar rats (3/step) were given a single oral dose of N1,N3-diallylpropane-1,3-diamine dihydrochloride (100% purity) in water at levels of 300 and 2000 mg/kg bw. Animals were then observed for 14 days.

At a dose level of 300 mg/kg bw, there were neither mortalities nor clinical findings of toxicological relevance. However, 3 out of 6 treated animals at this dose level showed a slight piloerection 1-2 hours after test item administration. All animals completely recovered within 3 hours and therefore these findings are assumed to be a sign of discomfort to the test item administration rather than a toxicological effect. No specific gross pathological changes were recorded for surviving animals.

After oral administration of 2000 mg/kg bw, two animals died spontaneously on day 1 and day 3 after treatment. One animal had to be sacrificed for ethical reasons on day 3 after treatment. The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, hunched posture, piloerection and apathy. The clinical signs persisted until the premature end of the observation period in all animals. Necropsy of the animals, which were found dead showed the beginning of autolysis, necropsy of the animal which had to be sacrificed for ethical reasons revealed diffuse red discoloured, fluid filled lungs. There were no treatment related changes in body weight at either dose level.

In accordance with OECD TG 423, the LD50 cut-off value for N1,N3-diallylpropane-1,3-diamine dihydrochloride after a single oral administration is considered to be 500 mg/kg bw.

This acute oral study is classified as acceptable. It does satisfy the guideline requirement for an acute oral study (OECD 423) in the rat.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

500 mg/kg bw

Quality of whole database:

Study is a GLP-compliant Guideline study with Klimisch score 1.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-04-16 to 2020-08-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity: Fixed Dose Procedure)

Version / remarks:

adopted 09th October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was used as delivered by the sponsor. In order to ensure good skin contact, it was moistened with 1 mL of aqua ad injectionem (Deltamedica, lot 910137, expiry date: September 2022). This vehicle was chosen due to its non-irritating characteristics.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9-12 weeks
- Weight at study initiation: 217-232 g
- Housing: The animals were kept in groups except during exposure and unless there were reasons to house individually (e.g. if there is concern that contact with other animals could increase stress due to the nature and severity of the signs of toxicity, or could result in exacerbation of local skin effects) in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: Tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: Adequate acclimatisation period (at least five days) under laboratory conditions
- Microbiological status when known : The animals were derived from a controlled full-barrier maintained breeding system (SPF).

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

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage: The test item was applied at a single dose, uniformly over an area which was approximately 10% of the total body surface.
- Type of wrap if used: The test item was held in contact with the skin by a dressing throughout a 24-hour period. This consisted of a semi-occlusive dressing made of a porous gauze and non-irritating tape and was fixed with an additional dressing in a suitable manner.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period the residual test item was removed using aqua ad injectionem (sterile water).
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied: 2000 mg/kg bw
- For solids: The test item was used as delivered by the sponsor. In order to ensure good skin contact, it was moistened with 1 mL aqua ad injectionem.

Duration of exposure:

24 hours

Doses:

- 2000 mg/kg bw

No. of animals per sex per dose:

3 females

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: Signs of erythema and oedema were assessed using the scoring system laid down in OECD Guideline 404 (for details see Table 1 in box "Any other information on materials and methods incl. tables") at 24, 48 and 72 hours after patch removal.
- Frequency of weighing: The animals were weighed on day 1 (prior to the application), on days 8 and 15.
- Necropsy of survivors performed: At the end of the observation period the animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally at a dosage of 250–400 mg/kg bw. All animals were subjected to gross necropsy and examined macroscopically for gross pathological changes. In absence of gross pathological changes no tissues were preserved for a possible histopathological evaluation
- Clinical signs: A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 6 hours post-dose). As soon as symptoms were noticed they were recorded. Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded. Cageside 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. Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

n.a.

Preliminary study:

One animal was administered a dose of 2000 mg/kg bw. As the animal survived without showing any signs of systemic toxicity, this dose was used as a starting dose for the main study.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed after treatment with the test material.

Clinical signs:

other: No clinical signs were observed after treatment with the test material during the whole observation period.

Gross pathology:

No specific gross pathological changes were recorded for any animal.

Other findings:

No signs of acute dermal toxicity were observed after treatment with the test material during the whole observation period, but mild signs of irritation were observed in the main experiment which were fully reversible after Day 3 or Day 4 after patch removal (for details see Table 2 in section "Any other information on results incl. tables").

Table 2: Skin Irritation at Application Site - Individual Data – DRF and Main Study

Time after Patch Removal	Dose Range Finding Study		Main Study
	Animal No. 1*		Animal No. 2**		Animal No. 3**
	E/O	C	E/O	C	E/O	C
Day 2 (0±2 h)	0/0	nsf	1°/0	nsf	1°/0	nsf
Day 3 (24±2 h)	0/0	nsf	1°/0	nsf	1°/0	nsf
Day 4 (48±2 h)	0/0	nsf	0/0	nsf	1°/0	nsf
Day 5 (72±2 h)	0/0	nsf	0/0	nsf	0/0	nsf
Day 6 - 15	0/0	nsf	0/0	nsf	0/0	nsf

Table 2: Absolute Body Weights [g] and Body Weight Change [%] - DRF and Main Study

Animal No.	Absolute Body Weights [g]			Body Weight Change [%]
	Day 1	Day 8	Day 15	Day 1-15
1*	217	226	236	9
2**	223	225	231	4
3**	232	224	240	3

* = dose range finding study animal; ** = main study animal

Interpretation of results:

GHS criteria not met

Conclusions:

In an acute dermal toxicity study conducted according to OECD 402, N1,N3-diallylpropane-1,3-diamine dihydrochloride was dermally administered to rats for 24 hours. The application was associated with no mortality and neither signs of toxicity, except for mild signs of irritation which were reversible within 5 days. Based on the results, the dermal LD50 can be considered to be greater than 2000 mg/kg bw.

Executive summary:

In an acute dermal toxicity study conducted according to OECD 402, three female Wistar rats were dermally exposed to N1,N3-diallylpropane-1,3-diamine dihydrochloride (100% purity) under semi-occlusive conditions for 24 hours to approximately 10% of the total body surface at a dose of 2000 mg/kg bw. Animals were then observed for 14 days. There were no treatment related mortalities, no changes in body weight, no clinical signs or other signs of acute dermal toxicity, except mild signs of dermal irritation which were fully reversible within five days. Based on the results, the dermal LD50 can be considered to be greater than 2000 mg/kg bw and no classification according to CLP Regulation 1272/2008 is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

Suitable data is available for the substance N1,N3-diallylpropane-1,3-diamine dihydrochloride from Acute Oral and Dermal Toxicity studies conducted according to OECD TG 423 and 402.

In an acute oral toxicity study conducted according to OECD 423 (acute toxic class method), groups of female Wistar rats (3/step) were given a single oral dose of N1,N3-diallylpropane-1,3-diamine dihydrochloride (100% purity) in water at levels of 300 and 2000 mg/kg bw. Animals were then observed for 14 days.

At a dose level of 300 mg/kg bw, there were neither mortalities nor clinical findings of toxicological relevance. However, 3 out of 6 treated animals at this dose level showed a slight piloerection 1-2 hours after test item administration. All animals completely recovered within 3 hours and therefore these findings are assumed to be a sign of discomfort to the test item administration rather than a toxicological effect. No specific gross pathological changes were recorded for surviving animals.

After oral administration of 2000 mg/kg bw, two animals died spontaneously on day 1 and day 3 after treatment. One animal had to be sacrificed for ethical reasons on day 3 after treatment. The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, hunched posture, piloerection and apathy. The clinical signs persisted until the premature end of the observation period in all animals. Necropsy of the animals, which were found dead showed the beginning of autolysis, necropsy of the animal which had to be sacrificed for ethical reasons revealed diffuse red discoloured, fluid filled lungs. There were no treatment related changes in body weight at either dose level. In accordance with OECD TG 423, the LD50 cut-off value for N1,N3-diallylpropane-1,3-diamine dihydrochloride after a single oral administration is considered to be 500 mg/kg bw.

In an acute dermal toxicity study conducted according to OECD 402, three female Wistar rats were dermally exposed to N1,N3-diallylpropane-1,3-diamine dihydrochloride (100% purity) under semi-occlusive conditions for 24 hours to approximately 10% of the total body surface at a dose of 2000 mg/kg bw. Animals were then observed for 14 days. There were no treatment related mortalities, no changes in body weight, no clinical signs or other signs of acute dermal toxicity, except mild signs of dermal irritation which were fully reversible within five days. Based on the results, the dermal LD50 can be considered to be greater than 2000 mg/kg bw.

Justification for classification or non-classification

Based on the available study results, classification of N1,N3-diallylpropane-1,3-diamine dihydrochloride as Acute Tox 4, H302 is warranted in accordance with CLP Regulation 1272/2008.

No classification according to CLP criteria is warranted for the acute dermal toxicity endpoint since the LD50 value for the dermal route is above the limit value of the relevant OECD guideline.