Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates

Endpoint summary

Description of key information

No study is available for Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates. Subchronic toxicity studies are available for :

(Z)-1-(octadec-9-enylammonio)propane-3-ammonium diacetate (CAS 7173-67-3):
- 14d range-finder (GLP): LOAEL = 7 mg/kg bw/day (corresponding to 5 mg/kg bw/day in terms of oleyl diamine)

(Z)-N-9-octadecenyl-1,3-diaminopropane (CAS 7173-62-8):
- 14d range-finder (GLP): NOAEL = 2 mg/kg bw/day
- 14d study (GLP): LOAEL = 12.5 mg/kg bw/day
- 28d (OECD 407, GLP): NOAEL = 1.25 mg/kg bw/day

 N-C12,14 alkyl-1, 3-diamino propane (CAS 90640-43-0):
- Key study : 90d (OECD 408, GLP) - NOAEL = 0.4 mg/kg bw/day (corresponding to 0.7 mg/kg bw of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-05-14 - 2010-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier S.A.S, Route des Chênes secs-B.P.4105-53941 LE GENEST-ST-ISLE-France
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: Main study groups
Females: 177-237 g, (mean: 200.84 g, ± 20%= 160.67 -241.01g)
Males: 246-304 g, (mean: 277.28 g, ± 20%= 221.82- 332.74g)
Recovery groups (28 and 90 day recovery)
Females: 178-218 g, (mean: 196.45 g, ± 20%= 157.16 -235.74g)
Males: 250-289 g, (mean: 267.25 g, ± 20%= 213.80-320.70g)
- Housing: housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in corn oil. The vehicle was chosen due to its non-toxic characteristics as well as according to sponsor`s request. The test item formulation was prepared freshly on each administration day before the administration procedure.

VEHICLE
- Lot/batch no. (if required): 117KO127, 058KO 070 and 128KO 040 (Sigma)

Application volume: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed.
Samples for homogeneity were taken from the top, middle and bottom of the High Dose and Low Dose preparation. The determination was performed in week 1, 5 and 13 of the study. Analysis of the dose formulations of the test item in the vehicle (nominal concentration) was performed in week 1, 5, 9 and 13 of the study for all doses. The dose formulation analysis was performed at BSL Scientific Laboratories GmbH under the BSL Project Nr. 081585.

Duration of treatment / exposure:

up to 90-91 days. The animals in the recovery group were dosed for 90 days and further held for 28 and 90 days post dose treatment period.

Frequency of treatment:

single dose daily, 7 days per week for a period of 90-91 days

Dose / conc.:

0.1 mg/kg bw/day (actual dose received)

Dose / conc.:

0.4 mg/kg bw/day (actual dose received)

Dose / conc.:

1.5 mg/kg bw/day (actual dose received)

Dose / conc.:

6 mg/kg bw/day (actual dose received)

Remarks:

including recovery Groups (28 and 90 days)

No. of animals per sex per dose:

10 (Main Study); 5 (Recovery Groups)

Control animals:

yes, concurrent vehicle

Details on study design:

Additional information for dosage:
According to the results of the dose range finding study (BSL Study Number 081579) and the 28-Day Repeated Dose Toxicity Study (BSL Study Number 084079) and in consultation with the sponsor the following doses were selected:
Control: 0 mg/kg bw
LD: 0.1 mg/kg bw
MD: 0.4 mg/kg bw
HID: 1.5 mg/kg bw
HD: 6 mg/kg bw
Recovery Groups:
CR: 0 mg/kg bw
HDR: 6 mg/kg bw
The highest dose level was chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and no-observed-adverse effects at the lowest dose level (NOAEL). The animals in the control group were handled in an identical manner to the dose group subjects and received the vehicle in the same volume.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection). The observation period was 90-91 days (main study group), 118 days (28-Day recovery group) and 180 days (90-Day recovery group).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made once prior to initiation of the treatment for all animals (main and recovery groups) and weekly thereafter. Once before the first exposure and once in the last week of treatment in all animals (main and recovery groups) and in the 4th week of recovery (28-day recovery groups) and in the last week of recovery (90-day recovery groups), sensory reactivity to stimuli of different types was conducted with specific emphasis on locomotion and behaviour. These observations were made outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight for all animals was measured at randomisation, daily during treatment period and on the day of terminal sacrifice. In recovery animals, after the treatment period, body weight was measured weekly during recovery period.

FOOD CONSUMPTION: Yes
- Food consumption was measured weekly during the treatment period for all animals and weekly during recovery period for recovery animals.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once prior to administration of the test article in all animals and once at the terminal sacrifice of main study animals (week 0 and 12).
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood samples were collected at terminal sacrifice (main and recovery group animals)
- Animals fasted: Yes (overnight)
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: clinical biochemistry determinations were performed on serum samples obtained from all animals as a part of the procedure of killing the animals for necropsy
- Animals fasted: Yes (overnight)
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was performed on the samples collected from main and recovery group animals at terminal sacrifice.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: All animals (main and recovery groups) of the study were subjected to detailed gross necropsy on day 91-92 for main group animals and day 119 and 181 for recovery group animals. During necropsy, a careful examination of the external surface of the body, all orifice and the cranial, thoracic and abdominal cavities and their contents was made. The wet weight of the following organs was taken in all terminally sacrificed animals as soon as possible. Paired organs were weighed separately: liver, ovaries, kidneys, uterus with cervix, adrenal glands, thymus, testes, spleen, epididymides, brain, prostate, heart, and seminal vesicles with coaulating glands

HISTOPATHOLOGY: Yes: The following tissues from all animals were preserved in 10% neutral buffered formalin except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative: all gross lesions, heart, aorta, skin, brain, ovaries, spinal cord, uterus with cervix, eyes, vagina, liver, testes, kidneys, epididymides, adrenal glands, prostate, stomach, seminal vesicles with coaulating glands, small and large intestines, urinary bladder, thymus, lymph nodes, thyroid gland, peripheral nerve, spleen, bone marrow, lungs and trachea, bone and skeletal muscle, pituitary, oesophagus, pancreas, salivary glands, parathyroids, mammary gland, nasal cavity, and larynx.
The afore-listed organs were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Full histopathology was carried out on the preserved organs and tissues of all animals in the control and high dose groups (main groups). These examinations were extended to animals of all other dosage groups as treatment-related changes were observed in the high dose group. All gross lesions were examined histopathologically.
For the recovery groups, histopathology was performed on tissues and organs identified as showing effects in the main groups. Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, GB - Hereford HR2 6JU. Histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Other examinations:

Inflammatory Marker
Serum samples collected at terminal sacrifice (main and recovery groups) have been stored at = -20 °C for analysis of inflammatory markers by ELISA technique. The analysis results will be reported separately.

Statistics:

For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test or unpaired t test were carried out to reveal any differences between control- and test groups. The statistical analysis was performed with GraphPad Prism V.x software (p < 0.05 was considered as statistical significant).
In the evaluation of laboratory parameters, all values within a range of the mean value ± the two fold standard deviation (x ± 2s) are considered to be „normal“ values within a „normal“ population

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item specific clinical signs were observed in any of the animals of LD and MD of main group. The predominant clinical signs observed in HID and HD main and recovery group animals were slight to severe salivation, half-eyelid closure, vocalization, temporary stertorous respiration, oral and nasal discharge, dyspnoea, weight loss and piloerection.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three mortalities in the main groups were observed (2 female HD and 1 male HID). There were also 2 mortalities in high dose females of HDR-90 group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In main study males, HD group animals showed statistically significant decrease in overall body weight gain (day 1 - 90) as compared to corresponding controls. Body weight gain values from LD, MD and HID were comparable with that of corresponding control group. In females, all treated group animals showed marginal decrease in overall body weight gain (day 1-90) as compared to corresponding controls. However, this effect was not statistically significant.
In 28-day recovery group males, the HD animals showed a statistically significant decrease in overall body weight gain during the treatment period (day 1-90) as compared to controls. However, body weight gain during 28-day recovery period was increased as compared to controls. In 28-day recovery group female animals, overall body weight gain during the treatment period (day 1 - 90) and 28-day recovery period was comparable to corresponding controls.
In 90-day recovery group males, the HD animals showed a decrease in overall body weight gain during the treatment period (day 1 - 90) as compared to controls without reaching statistical significance. However, body weight gain during 90-day recovery period was increased as compared to controls. In 90-day recovery group female animals, there was no any effect on body weight during treatment and recovery period.
The difference in weight gain among the groups could be attributed to the treatment with test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In main study males, HD group animals showed statistically significant decrease in food consumption (day 1 - 90) as compared to corresponding controls. Food consumption from LD, MD and HID was slightly decreased without reaching statistical significance when compared with that of corresponding control group. In females, all treated group animals showed decrease in food consumption (day 1 - 90) as compared to corresponding controls. However, this effect was not statistically significant. In 28-day recovery group males, the HD animals showed a decrease in food consumption during the treatment period (day 1 - 90) as compared to controls without achieving statistical significance. However, food consumption during 28-day recovery period was slightly increased as compared to controls. In 28-day recovery group female animals, food consumption during the treatment period (day 1 - 90) and 28-day recovery period was comparable to corresponding controls. In 90-day recovery group males, the HD animals showed a decrease in food consumption during the treatment period (day 1 -90) as compared to controls without reaching statistical significance. However, food consumption during 90-day recovery period was increased as compared to controls. In 90-day recovery group female animals, there was no significant effect on food consumption during treatment and recovery period. The difference in food consumption among the groups could be attributed to the treatment with test item as it was correlated with body weight decrease during the respective period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The blood analysis for haematology and clinical pathology parameters revealed most of the mean and individual values within the biological range. Statistically significant differences in few parameters were found between the treated groups and the corresponding control group.
For haematology, except for Hct values in MD and HID males and MD, HID and HD females, RBC in main study males of MD group and females of HDR-28, MCV in HDR-28 females, MCH in HDR-28 females, MCHC in MD and HDR- 28 males, WBC in HD males, no other parameters showed any significant difference compared to controls. Individual values for most of the parameters were within the biological range.
In most of the main and recovery group males and females, the mean and individual PTT and aPTT values were below the biological range. Statistically significant decrease in HD main study males were observed for PTT when compared to the controls.
Statistically significant decrease in aPTT was observed in LD, MD, HID males, LD, MD females and HDR-90 males when compared with respective controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For clinical biochemistry, except for AST/GOT in HD males, TP in female HID and HD group, GLU in HDR-90 males, UREA in female HID, no other parameters showed any significant deviation compared to controls. No treatment related effect on urine parameters were found in treated groups as compared to control group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No relevant differences between test and control groups were found for both main and recovery animals. No dose dependency and toxicological relevance was observed in various urine parameters.
Low pH values (acidic urine, pH 5-6) were observed in few animals.
Main study groups: 6/10 male and 9/10 female control; 6/10 male and 7/10 female of LD group; 2/10 male and 7/10 female MD group; 5/10 male and 6/10 female HID group; 10/10 male and 8/8 female HD group.
28 day recovery groups: 2/5 male and 4/5 female control; 5/5 male and 3/3 female HD group.
90 day recovery groups: 2/5 male and 3/5 female control; 2/5 male and 3/3 female HD group.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No effect on organ weights was observed except statistically significant decrease in absolute liver weight in HD males, increase in relative kidney weights in HDR-28 males, increase in relative adrenal weights in male and female HD, increase in relative heart weights in males of MD group, decrease in absolute brain weights in HID males, increase in relative brain weights in HD males and decrease in absolute brain weights in HDR-28 males and increase in relative epididymitis weights in HD males.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At terminal sacrifice of main study and recovery animals, macroscopic findings observed were considered spontaneous and not treatment related.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were seen in the small intestine (ileum and jejunum), mesenteric lymph node, spleen, bone marrow (sternum) and trachea of terminally sacrificed animals.
After the 28-day recovery period, in animals of the HDR-28 dose group, test item-related changes in the jejunum, ileum and mesenteric lymph node were still present. Changes noted in the spleen had partially regressed and bone marrow (sternum) and trachea changes had fully regressed.
After 90 days of recovery, in animals of the HDR-90 dose group, test item-related changes in the jejunum, ileum and mesenteric lymph node had partially regressed. No test item-related changes were noted in the spleen, bone marrow (sternum) and trachea.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

0.4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

Critical effects observed:

not specified

Table 1: Haematology data

Group	Parameter	Hct	RBC	MCV	MCH	MCHC	WBC	PTT	aPTT
	Unit	%	x10^6/µl	fL	pg	g/dL	x10^3/µl	sec	sec
	Biol. Range	28.3-54.9	6.3-10.54	45.5-60.8	15.4-19.8	30.7-36.7	2.9-25	29-51	10.9-37
Male
C	Mean SD N	49.05 1.5 10	9.07 0.37 10	54.11 1.55 10	17.03 0.71 10	31.5 0.6 10	10.63 2.06 10	27.61 5.36 10	10.69 3.44 10
LD	Mean SD N	50.95 3.47 10	9.33 0.64 10	54.63 1.51 10	17.07 0.6 10	31.24 0.59 10	10.53 3.05 10	20.61* 5.06 10	11.28 2.34 10
MD	Mean SD N	53.72* 2.53 8	9.83* 0.5 8	54.66 0.76 8	16.73 0.35 8	30.58* 0.61 8	10.01 0.74 8	19.98* 4.43 8	10.95 2.12 8
HID	Mean SD N	52.87* 4.11 9	9.59 0.76 9	55.15 1.03 9	17.06 0.49 9	30.92 0.58 9	9.33 2.36 9	17.32* 3.2 9	9.75 1.37 9
HD	Mean SD N	49.71 2.78 10	9.31 0.36 10	53.37 1.49 10	16.74 0.63 10	31.37 0.83 10	16.19* 6.77 10	25.44 6.42 10	11.35 2.22 10
CR28	Mean SD N	48.96 2.63 5	9.01 0.65 5	54.44 2.14 5	17.58 0.82 5	32.29 0.54 5	11.26 2.05 5	21.12 1.95 5	11.49 0.88 5
HDR28	Mean SD N	52.99 3.39 5	9.94 0.71 5	53.33 1.34 5	16.54 0.68 5	31.01* 0.81 5	13.32 3.06 5	23.39 3.16 5	12.26 2.58 5
CR90	Mean SD N	48.69 2.0 5	8.74 0.57 5	55.76 1.92 5	18.06 1.07 5	32.4 0.84 5	9.46 1.92 5	45.99 4.67 5	15.19 2.41 5
HDR90	Mean SD N	49.49 2.28 5	8.98 0.41 5	55.11 1.04 5	17.47 0.51 5	31.69 0.77 5	10.76 3.54 5	34.28* 4.11 5	13.1 0.41 5
Female
C	Mean SD N	43.92 1.32 10	7.93 0.41 10	55.47 1.29 10	18.12 0.7 10	32.66 0.8 10	5.46 2.22 10	22.4 11.9 10	11.15 3.73 10
LD	Mean SD N	45.58 1.5 10	8.28 0.27 10	55.07 0.36 10	18.0 0.45 10	32.69 0.78 10	5.59 0.84 10	13.6* 5.38 10	8.66 3.1 10
MD	Mean SD N	46.84* 2.46 10	8.42 0.4 10	55.62 1.74 10	17.84 0.43 10	32.08 0.53 10	4.96 1.2 10	14.51* 1.23 10	9.31 1.39 10
HID	Mean SD N	46.35* 2.83 10	8.37 0.51 10	55.41 0.81 10	17.7 0.37 10	31.94 0.7 10	5.17 2.28 10	16.9 2.77 10	9.65 1.12 10
HD	Mean SD N	46.66* 1.08 8	8.38 0.33 8	55.71 1.74 8	17.9 0.44 8	32.14 0.58 8	7.44 2.38 8	19.11 4.44 8	10.49 1.44 8
CR28	Mean SD N	45.89 1.24 5	8.14 0.1 5	56.34 0.94 5	18.48 0.57 5	32.76 0.6 5	4.39 2.26 5	24.88 5.23 5	12.91 1.5 5
HDR28	Mean SD N	46.46 1.72 5	8.52* 0.3 5	54.5* 0.41 5	17.66* 0.5 5	32.36 0.82 5	5.32 1.77 5	25.24 2.45 5	12.44 1.05 5

Table 2: Clinical biochemistry data

Group	Parameter	AST/GOT	TP	GLU	UREA
	Unit	U/L	g/L	mmol/L	mmol/L
Male
C	Mean SD N	71.39 73.45 10	55.85 2.93 10	13.17 2.63 10	6.0 0.95 10
HD	Mean SD N	40.23 12.91 10	53.25 4.56 10	13.05 1.03 10	6.81 0.84 10
CR90	Mean SD N	44.36 3.09 5	59.34 0.97 5	14.74 1.23 5	6.91 0.65 5
HDR90	Mean SD N	139.02 193.73 5	57.66 1.35 5	12.23* 1.76 5	7.14 1.02 5
Female
C	Mean SD N	41.48 11.35 9	58.81 4.48 9	8.89 1.2 9	6.78 0.46 9
HID	Mean SD N	30.62 4.42 10	55.21* 2.96 10	9.88 2.48 10	5.57* 0.79 10
HD	Mean SD N	33.63 6.1 8	53.08* 2.55 8	9.92 1.25 8	6.01 1.11 8

*Significant (p<0.05); as determined with the individual data

Conclusions:

Test item related clinical signs were observed in HID and HD main groups and HD recovery groups. Predominant signs observed were reduced spontaneous activity, salivation, nasal discharge and moving the bedding.

Three mortalities in the main groups, (two female from HD and one male from HID) and 2 mortalities in high dose females of HDR-90 group were observed.

In main study and recovery HD group male and female animals, there was statistically significant decrease in overall body weight gain (day 1-90) and food consumption was observed compared to corresponding controls. Body weight gain and food consumption values from MD and HID were slightly decreased without achieving a statistical significance when compared with control. However, these parameters remained unaffected in low dose and values were comparable with the controls.

No test item related effect in any of the haematology parameters, urine parameters and clinical biochemistry parameters in male and females of any group except decrease in TP in HD females was observed.

At terminal sacrifice of main study and recovery animals, macroscopic findings observed were considered to be spontaneous and not treatment related

No effect on organ weight in male and female animals of treated group and recovery group was observed which could be considered as a treatment related effect except decrease in absolute liver weights in HD males, increase in relative kidney weights in HDR-28 males, increase in relative adrenal weights in HD male and females and increase in relative epididymides weight in HD males.

Histopathologically, test item-related changes were seen in the small intestine (ileum and jejunum), mesenteric lymph node, spleen, bone marrow (sternum) and trachea of the MD, HID and HD animals. After the 28-day recovery period, item-related changes in the jejunum, ileum and mesenteric lymph node were still present in animals of the HDR-28 dose group. Changes noted in the spleen had partially regressed and bone marrow (sternum) and trachea changes had fully regressed.
After 90 days of recovery, in animals of the HDR-90 dose group, test item-related changes in the jejunum, ileum and mesenteric lymph node had partially regressed.
No test item-related changes were noted in the spleen, bone marrow (sternum) and trachea.
All findings observed in this 90 day study are comparable with 28 day repeated dose toxicity study performed with N-C12,14 alkyl-1,3-diaminopropane (BSL Study No. 084079). It is indicative that the observed effects are local and might have induced by inflammatory changes and therefore treatment related observed effects in various parameters could be secondary changes to the inflammatory responses.
This 90 day repeated dose oral toxicity study including a 28 and 90 day recovery period revealed treatment related changes in few parameters of high dose group male and females. Except histopathology, slight effect on body weight, food Report BSL consumption and clinical signs, no test item related effect was observed in any of the parameters evaluated in MD and HID group. Based on these findings and the histopathological evaluation, the NOAEL (No-Observed-Adverse-Effect-Level) was considered to be 0.4 mg/kg/day under the circumstances of this study.

Executive summary:

In this 90 day repeated dose oral toxicity study (performed according to the First Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 407, “Repeated Dose 28-day Oral Toxicity Study in Rodents” adopted 03 October 2008, First Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 408, “Repeated Dose 90 day Oral Toxicity Study in Rodents” adopted 21 September 1998, Directive 96/54 EEC B.7 and Directive 2001/59/ EC B.26), the test item N-C12,14 alkyl-1,3-diaminopropane was suspended in corn oil and orally administered in graduated doses to four groups of male and female rats (Wistar RjHan:WI) by oral gavage, using a gavaging canula. The main study included 5 groups (C, LD, MD, HID, HD) with each comprised of 10 male and 10 female animals. 28 day and 90 day recovery study included 2 groups (CR-28, HDR-28, CR-90 and HDR-90) each with 5 male and 5 female animals. 

Total 90 applications per animal were administered and the animals of the recovery groups were kept for further 28 and 90 days following the last administration before terminal sacrifice. 

The following doses were chosen: Low Dose: 0.1 mg/kg bw, Medium Dose: 0.4 mg/kg bw, High Intermediate 1.5 mg/kg bw and High Dose: 6 mg/kg bw. The volume of application was 4 mL/kg bw.

All surviving animals were sacrificed on day 91 – 92 (main group animals) day 119 and 181 (recovery groups). 

Clinical signs and Mortality

No test item specific clinical signs were observed in any of the animals of control LD, MD of main group. The predominant clinical signs observed in HID and HD main and recovery group animals were slight to severe salivation, half-eyelid closure, vocalization , temporary stertorous respiration, oral and nasal discharge, dyspnoea, weight loss and piloerection.

Three mortalities in the main groups were observed ( two female HD and one male HID).There were also 2 mortalities in high dose females of HDR-90 group.

 

Body Weight

In main study males, HD group animals showed statistically significant decrease in overall body weight gain (day 1-90) as compared to corresponding controls. In females, all treated group animals showed marginal decrease in overall body weight gain (day 1-90) as compared to corresponding controls. However, this effect was neither dose related nor statistically significant.

In 28 day recovery group males, the HD animals showed a statistically significant decrease in overall body weight gain during the treatment period (day 1-90) as compared to controls. However, body weight gain in during 28 day recovery period was increased as compared to controls. In 28 day recovery group female animals, overall body weight gain during the treatment period (day 1-90) and 28 day recovery period was comparable to corresponding controls. 

In 90 day recovery group males, the HD animals showed a decrease in overall body weight gain during the treatment period (day 1-90) as compared to controls without reaching statistical significance. However, body weight gain during 90 day recovery period was increased as compared to controls. In 90 day recovery group female animals, there was no any effect on body weight during treatment and recovery period.

 

Food Consumption

In main study males, HD group animals showed statistically significant decrease in food consumption (day 1-90) as compared to corresponding controls. Food consumption from LD, MD and HID was slightly decreased without reaching statistical significance when compared with that of corresponding control group. 

In females, all treated group animals showed decrease in food consumption (day 1-

90) as compared to corresponding controls. However, this effect was not statistically significant. 

In 28 day recovery group males, the HD animals showed a decrease in food consumption during the treatment period (day 1-90) as compared to controls without achieving statistical significance. However, Food consumption during 28 day recovery period was slightly increased as compared to controls. In 28 day recovery group female animals, food consumption during the treatment period (day 1-90) and 28 day recovery period was comparable with corresponding controls. In 90 day recovery group males, the HD animals showed a decrease in food consumption during the treatment period (day 1-90) as compared to controls without reaching statistical significance. However, food consumption during 90 day recovery period was increased as compared to controls. In 90 day recovery group female animals, there was no significant effect on food consumption during treatment and recovery period.  

 

FOB

No differences were observed concerning functional and behavioral examination prior to application and during the last week of treatment. No treatment related abnormalities were recorded concerning posture, gait, palpebral closure, lacrimation, piloerection, arousal and vocalization. No convulsions, tremors, stereotypy or bizarre behaviour were observed in any of the animals.

 

Haematology and Clinical Biochemistry

The blood analysis for haematology and clinical pathology parameters revealed most of the mean and individual values within the biological range. Statistically significant differences in few parameters were found between the treated groups and the corresponding control group. 

 

For hematology, except for Hct values in MD and HID males and MD, HID and HD females, RBC in in main study males of MD group and females of HDR-28, MCV in in HDR-28 females, MCH in HDR-28 females, MCHC in MD and HDR- 28 males, WBC in HD males, no other parameters showed any significant difference compared to controls. Individual values for most of the parameters were within the biological range.

 

Effect on clotting parameters like PTT and aPTT can not be attributed to the treatment with test item and considered to be an incidental due to lack of dose dependency and majority of the group mean values were within the biological range. 

 

Statistically significant decrease in aPTT was observed in LD, MD, HID males, LD, MD females and HDR-90 males when compared with respective controls. 

 

For clinical biochemistry, except for AST/GOT in HD males, TP in female HID and HD group, GLU in HDR-90 males, UREA in female HID, no other parameters showed any significant deviation compared to controls.   

No treatment related effect on urine parameters were found in treated groups as compared to control group.

 

Gross Pathology

At terminal sacrifice of main study and recovery animals, macroscopic findings observed were considered to be spontaneous and not treatment related.

 

Organ Weight

No effect on organ weights was observed except statistically significant decrease in absolute liver weight in HD males, increase in relative kidney weights in HDR-28 males, increase in relative adrenal weights in male and female HD, increase in relative heart weights in males of MD group, decrease in absolute brain weights in HID males, increase in relative brain weights in HD males and decrease in absolute brain weights in HDR-28 males and increase in relative epididymides weights in HD males.

 

Histopathology

Test item-related changes were seen in the small intestine (ileum and jejunum), mesenteric lymph node, spleen, bone marrow (sternum) and trachea of terminally sacrificed animals.

 

After the 28-day recovery period, in animals of the HDR-28 dose group, test item-related changes in the jejunum, ileum and mesenteric lymph node were still present. Changes noted in the spleen had partially regressed and bone marrow (sternum) and trachea changes had fully regressed.

 

After 90 days of recovery, in animals of the HDR-90 dose group, test item-related changes in the jejunum, ileum and mesenteric lymph node had partially regressed. No test item-related changes were noted in the spleen, bone marrow (sternum) and trachea.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.7 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

90-day study (OECD 408) sufficient to cover Annex IX requirements. Study is GLP compliant with Klimisch score 1. The applied read-across is adequate, and results are consistent with other available data in the diamine category.

System:

gastrointestinal tract

Organ:

intestine

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The fatty diamine salts are produced by simple mixing of the corresponding diamines with acetic acid under strictly controlled conditions to avoid by-product formation. Thus, the resulting acetates are expected to have comparable toxicity profiles like their diamine precursors when dissolved in water or physiological media without influence of the acetate group. Regarding the toxicity of the fatty diamines, it is expected that the members with a shorter chain and/or a higher degree of unsaturation will have a higher toxicity among the category or at least will represent the worst case. Thus, a read-across could be applied to longer chains into shorter and to saturated molecules into unsaturated ones.

 

As for the diamines family, grouping could also be made for the fatty diamines salts as the only difference is related to the alkyl chain of the diamine part. For both salt substances, the anionic fatty acid part is acetate (mono or di-). The alkyl chain difference in the diamines used for both products mostly differ in the amount of unsaturation: for Tallow-DoAc average about 30-50 g I₂/100 g and > 70 g I₂/100 g for Oleyl-DoAc ((Z)-1-(octadec-9-enylammonio)propane-3-ammonium diacetate, CAS 7173-67-3). Both substances are considered comparable enough to use cross-reading. Based on the higher level of unsaturation of oleyl compared to tallow, the Oleyl-DoAc is considered to represent the worst case of the family and thus the data collected for the Oleyl_Diamine registration ((Z)-N-9-octadecenyl-1,3-diaminopropane , CAS 7173-62-8) or other shorter chain diamines as coco_diamine (N-C12,14 alkyl-1, 3-diamino propane; CAS 90640-43-0) will be used to assess the toxicity of the DoAc family.

 

Repeated dose toxicity by the oral route:

A total of four studies are available on (Z)-N-9-octadecenyl-1,3-diaminopropane. These included three 14-day studies (including one performed with the diacetate salt) and one 28-day study. An additional 90-day study was available with N-C12/14 alkyl-1, 3-diaminopropane, which is used for read across. All studies were performed according to OECD guidelines and under GLP conditions.

 

The 14-day dose range-finding study with (Z)-1-(octadec-9-enylammonio)propane-3-ammonium diacetate (Rudragowda 2009, 083754A) was performed with nominal dose groups of 0, 7, 17, 70 and 275 mg/kg bw/day (gavage: 10 ml/kg bw in water). All animals died in the highest dose group and there were clear signs of treatment-related effects (effects on bodyweight gain and food consumption, clinical chemistry, haematology, organ weights) in all dose groups. A NOAEL was not established based on these results. A LOAEL of 7 mg/kg/bw (corresponding to 5 mg/kg bw/day in terms of oleyl diamine content actually ingested) was established on the base of macroscopic effects in stomach, intestine, spleen and thymus.

 

The second 14-day dose range-finding study (Rudragowda, 2009, 081578) was performed on (Z)-N-9-octadecenyl-1,3-diaminopropanewith dose groups of 0, 2, 5, and 12.5 mg/kg/bw (gavage, corn oil as vehicle). A NOAEL of 2 mg/kg bw/day was determined primarily based on observations of enlarged lymph node adjoining the intestinal wall in females in the mid dose. Other findings in the highest dose group mirrored what was observed in the 14-day study with the diacetate form.

 

The third 14-day study (Rudragowda, 2009, 081564) was not a dose range-finding study but an investigation on the impact of a single administration of (Z)-N-9-octadecenyl-1,3-diaminopropane at 25 mg/kg bw/day against an administration of 12.5 mg/kg bw twice daily. The effects were similar to what was observed in the previous studies and a LOAEL of 12.5 mg/kg bw/day was established. There was no significant difference in toxicity following the two different administration schemes.

 

The 28-day study was performed on (Z)-N-9-octadecenyl-1,3-diaminopropanewith dose groups of 1.25, 5, and 20 mg/kg bw/day in corn oil administered per gavage. A 14-day recovery group was also included. There was an effect on bodyweight gain and organ weight (kidney, thymus, heart, brain, testes) primarily in the high dose group. Changes in haematological (possibly indicating an inflammatory response) and clinical chemistry parameters were observed. The NOAEL was set at 1.25 mg/kg bw/day primarily based on observations of enteropathy with foam cell accumulation in the small intestine and mesenteric lymph node with accompanying inflammatory changes seen in the mid and high dose groups. These changes were not reversible during the 14-day recovery period.

 

The 90-day study was performed onN-C12,14 alkyl-1, 3-diamino propane (CAS 90640-43-0) with dose groups of 0, 0.1, 0.4, 1.5 and 6 mg/kg bw/day (in corn oil) administered per gavage daily for 90 days. Additional groups of animals were administered 0 or 6 mg/kg bw/day for 90 days and were then observed for recovery over 28 or 90 days. There was mortality in the highest dose group but some of the deaths were attributed to gavage errors. However, it cannot be excluded that substance-related mortality was observed in the highest dose group. There were no effects in the two lowest dose groups and the NOAEL was set at 0.4 mg/kg bw/day. The main effects in highest dose group were on bodyweight gain (with recovery following end of dosing) and organ weights (liver, kidney, adrenal glands, epididymis). These changes were not accompanied by histological changes and they were specific to one sex (with the exception of adrenal gland). The toxicological relevance of these findings can be questioned. There were however other substance-related histological changes on the small intestine (ileum and jejunum), mesenteric lymph node, spleen, bone marrow and trachea. The histological changes in both the intestine and mesenteric lymph nodes were indicative of accumulation and infiltration of foam cells. The changes in the small intestine did regress during the recovery period but were still present after 90 days.

 

Consequently, the available subchronic toxicity studies indicate an effect on the small intestine and mesenteric lymph nodes with subsequent strong inflammatory response. This was observed with all the studies with (Z)-N-9-octadecenyl-1,3-diaminopropane (including the diacetate form) as well as that with N-C12,14 alkyl-1, 3-diamino propane that supports the current approach of grouping of substances and read-across of data. There were relatively minor differences in the effects on organ weights between the substances. These differences are likely to represent experimental performance uncertainties rather than a substance-related effect. When taking into consideration the relatively strong irritant/corrosive effects of these substances and the route of administration, it cannot be excluded that the overall toxicity reflects a point-of-first-contact effect. Thus for this endpoint, read-across from the 90 day study with N-C12,14 alkyl-1, 3-diamino propane is used for the evaluation of the diamine acetates compounds. This study indicated a NOAEL of 0.4 mg/kg bw/day based on the effects observed in the small intestine and the mesenteric lymph nodes.

 

Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates is a salt of N-(C16-18 and C18-unsatd. alkyl)trimethylenediamine and acetic acid, in a molar ratio of 1:2. Thus for DNEL setting use the 90-day oral NOAEL of 0.4 mg/kg for C12-14-diamine is therefore taken as starting point for the NOAEL value for tallow-diamine, which then is converted to the corresponding value for the diacetate salt of tallow-diamine.

(Mw C12 -14-diamine = 250, Mw tallow-diamine diacetate = 430; 0.4 x 430/250= 0.7 mg/kg bw/day)

Repeated dose toxicity by inhalation:
Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates is solid at room temperature or formulated as a liquid with pasty elements. The vapour pressure is less than 0.0015 Pa at 20°C (value is an overestimation as it is based on read-across from shorter chain C12-14-diamine). In addition, 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.

Repeated dose toxicity by the dermal route:

Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates is severely irritant to the skin and is not expected to easily pass the skin. The skin is therefore not a preferred route when studying repeated dose systemic toxicity.

Justification for classification or non-classification

Evaluation for classification is based on the available 90-day study on N-C12,14 alkyl-1, 3-diamino propane and is supported by data from cross-reading within the category of diamines from an available 28-day study on (Z)-N-9-octadecenyl-1,3-diaminopropane.

These available data indicate that all alkyl-diamines with alkyl chain lengths between C12 and C18 (represented by Oleyl) should be classified STOT RE 1; H372 in accordance with Regulation (EC) No 1272/2008:

- 90-day study on N-C12,14 alkyl-1, 3-diamino propane: This study showed significant toxicity at the highest tested dose of 6 mg/kg bw/day based on mortality observed in the highest dose group. This is below 10 mg/kg bw/day and therefore results to classification STOT RE 1; H372.

- 28-day study on (Z)-N-9-octadecenyl-1,3-diaminopropane: This resulted to significant toxicity (mortality) at the highest dose of 20 mg/kg bw/day with effects on weight of body and organs without histological changes. This is below 30 mg/kg bw and therefore also results to classification STOT RE 1; H372.