Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine

Endpoint summary

Description of key information

There is no study available on Oleyl-diamine mono-oleate itself. Read-across is applied to:

Oleyl-diamine dioleate:28 dayoral gavagerat (OECD 407, GLP): NOAEL 5 mg/kg bw/day

Oleyl-diamine: 28d oral gavage rat (OECD 407, GLP): NOAEL 1.25 mg/kg bw/day

C12-14-diamine: 28 day oral gavage rat (OECD 407, GLP): NOAEL 0.4 mg/kg bw/day

C12-14-diamine: 90 day oral gavage rat (OECD 408, GLP): NOAEL 0.4 mg/kg bw/day

No study available for inhalation or dermal exposure:

In the conservative approach that Oleyl-diamine is of equal toxicity as C12-14-alkyl-diamine, this leads to a 90-day NOAEL of 0.4 mg/kg bw/day for Oleyl-diamine, which is equivalent to 0.75 mg Oleyl-diamine mono-oleate/kg bw/day

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

Remarks:

Doses / Concentrations:
0.0, 0.1, 0.4, 1.5 and 6 mg/kg bw; Recovery Groups (28 and 90 days): 0 and 6 mg/kg bw
Basis:
actual ingested

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

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
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.
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.


BODY WEIGHT AND WEIGHT GAIN
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
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.


HAEMATOLOGY
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 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 CHEMISTRY
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
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.


ORGAN WEIGHTS
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.


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


HISTOPATHOLOGY: NON-NEOPLASTIC
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.

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:

histopathology: non-neoplastic

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:

LOAEL

0.4 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

7

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 likely to result to an overestimation of toxicity as testing is performed on the most toxic member of the group of diamines.

System:

gastrointestinal tract

Organ:

intestine

mesenteric lymph node

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

Mode of Action Analysis / Human Relevance Framework

The most significant treatment-related changes in all studies performed on diamines are effects on the small intestine and mesenteric lymph nodes. A relatively strong inflammatory reaction is also observed at high dose levels. These effects in the gastro-intestinal tract have consistently been observed in amine based surfactants.

A mode of action has not been established but it is possible to suspect the known corrosivity to be at least partially involved. The observed effects are local and they are by some interpreted as phospholipidosis, something commonly observed following treatment with cationic amphiphilic material, including marketed pharmaceuticals, and generally considered to be non-adverse. When taking into consideration the relatively strong corrosive effects of this substance, and for substances belonging to the same group of chemicals, and the route of administration, it cannot be excluded that the overall toxicity reflects a point-of-first-contact effect.

Phospholipidosis is a plausible mechanism. In physiological circumstances, the diamines have a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Noteworthy in this respect is that recent research shows that the log distribution coefficient for cationic surfactants between water and phospholipid are possibly several orders of magnitude higher than between water and oil. The complex of cationic surfactant and phospholipids are difficult to digest by the macrophages, and they accumulate with the lysosomes. Recent (unpublished) studies have shown that these cationic surfactants are all lysosomotropic, and scored positive for phospholipidosis inin vitrostudies with HepG2 cells.

Additional information

Oleyl-diamine mono-oleate is a salt of (Z)-N-9-octadecenylpropane-1,3-diamine (Oleyl-diamine, CAS 7173-62-8) and Oleic acid (CAS 112-80-1), which dissociates into its components in aqueous environment.

Following systemic absorption after oral dosing, no distinction can be made between Oleyl-diamine absorption from Oleyl-diamine mono-oleate or from dosing of Oleyl-diamine itself. Consequently, the evaluation of the individual components is just as relevant for the hazard evaluation as testing with the actual salt. Oleic acid, like other natural fatty acids, is considered to be of lower toxicity than Oleyl-diamine. Proof of this is found in the comparison of the results from comparably performed 28-day studies on Oleyl-diamine and Oleyl-diamine-dioleic acid (see below). This proofs that systemic toxicity from Oleyl-diamine dioleate is fully driven by its Oleyl-diamine content. The same can be expected to be the case for Oleyl-diamine mono-oleate.

The evaluation for repeat dose toxicity of Oleyl-diamine is based on series of highly reliable GLP studies performed over the category of alkyl-diamines. (Information on the applicability of the read-across from various diamines to Oleyl-diamine can be obtained from the document "Category polyamines - 20170518.pdf" added to IUCLID Ch. 13.). Specifically read-across is applied from N-C12/14 alkyl-1,3-diaminopropane, CAS 90640-43-0, as a worst-case representative of the category of alkyl-diamine.

 

The data from studies on C12-14-alkyl-diamine provides for a conservative NOAEL for Oleyl-diamine, which subsequently can be used to set a sub-chronic NOAEL forOleyl-diamine mono-oleate

 

ORAL

 

Oleyl-diamine dioleate:

- 28-dayoral gavage rat

The repeated dose toxicity of Oleyl-diamine dioleate was evaluated in 28-day oral gavage study in rats according to OECD 407 guideline and in compliance to GLP.

The selected dose levels were based on the results of a range finding/MTD study.

Four groups of 5 male and 5 female Wister rats received Oleyl-diamine dioleate dissolved in corn oil at dose levels of 0, 5, 20 and 100 mg/kg bw/day. Formulations were chemically analysed.

The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Effects at 100 mg: Clinical signs consisted of hunched posture, piloerection and lean appearance and lower motor activity. Body weights were lower than control (male 21%, females 14%) and at the clinical laboratory examinations WBC (neutrophils counts) was increased and albumin was decreased. Macroscopic examinations showed thickening and dilation of the jejunum, and reddish discolouration and enlargement of the mesenteric lymph nodes. Histological correlates of these findings were foamy macrophages foci present in the lamina propria of the duodenum, jejunum and/or ileum of both sexes. Moreover, (foamy) macrophage foci, granulocytic cell infiltrate and/or cellular debris, granuloma, granuloma with central necrosis, and lymphoid hyperplasia were noted in the mesenteric lymph nodes of male and female rats.

In general, macrophage foci present in the mesenteric lymph nodes may develop into more vacuolated (foamy) macrophages when taking up more material and finally being organized at higher doses into granuloma. In addition, granulocytes and cellular debris were present in these affected lymph nodes and in males some granuloma developed central necrosis.

At 20 mg/kg, foamy macrophage foci in the lamina propria of the jejunum in one female were observed. In addition, in the majority of the rats (foamy) macrophage foci, granulocytic cell infiltrate and/or cellular debris, granuloma and lymphoid hyperplasia were noted in the mesenteric lymph nodes.

At 5 mg/kg, foamy macrophage foci in the mesenteric lymph nodes was noted in one female only at a minimal degree. Given the incidental and minor nature of this finding, this was considered of no toxicological relevance.

Based on these results, a No Observed Adverse Effect Level (NOAEL) was established at the lowest dose level of 5 mg/kg.

 

Oleyl-diamine:

A total of 4 studies are available on Oleyl-diamine These included three 14-day studies (not included in this dossier) and one 28-day study. An additional 90-day study was available with N-C12/14 alkyl-1, 3-diaminopropane (C12-14-alkyl-diamine), which is used for read-across. All studies were performed according to OECD guidelines and under GLP conditions.

 

- 28-dayoral gavagerat

Oleyl-diamine (CAS 7173-62-8) has been evaluated for repeated dose toxicity in a 28-day repeated dose toxicity study according to OECD 407 guideline and in compliance to GLP. Four groups of five male and female rats were exposed by gavage to dose levels of 0, 1.25, 5, 20 mg Oleyl-diamine in corn oil/kg bw.

An additional group of control and high dose animals were subjected to the same treatment but with a 14 -day recovery.

In the high dose group there was one substance related death (prominent enteropathy of the small intestine with villous infiltration with foam cells and foam cells in mesenteric lymph nodes.). Further observations in the 20 mg/kg group were decreased BW gain, clinical signs (vocalization, piloerection, salivation, arched back, half eyelid closure, sneezing, reduced/increased spontaneous activity, diarrhoea and dyspnoea.), increased WBC (neutrophils) and decreased albumin. Furthermore, organ weights (absolute and relative) for brain, heart, thymus, testes and prostate showed differences to controls. All these effects had regressed in the recovery groups.

At 5 mg/kg clinical signs were observed of vocalization, increased spontaneous activity, nervousness and piloerection. Haematology showed increased WBC (neutrophils) and biochemistry mainly decreased albumin.

No test item specific clinical signs were observed in any of the animals of control and 1.25 mg/kg group

Histopathological examinations showed foam cell accumulations in small intestinal villi and mesenteric lymph node with accompanying inflammatory changes in small intestine, mesenteric lymph node and spleen essentially at 5 and 20 mg/kg/day, tracheal epithelial alteration and (multi)focal myocardial degeneration/fibrosis of the heart at 5 and 20 mg/kg/day, increased myeloid:erythroid ratio in the bone marrow at 20 mg/kg/day and foam cell accumulation in the thymus in a single animal at 20 mg/kg/day. Minimal foam cell accumulations in small intestinal villi were also observed in a single female at 1.25 mg/kg/day. After the recovery period, in animals of the 20 mg/kg/day dose group, foam cell accumulations in the small intestine and mesenteric lymph node had not regressed. Inflammatory intestinal, mesenteric lymph node and spleen changes had partially regressed.

The NOAEL was considered to be 1.25 mg/kg/day under the circumstances of this study.

 

The 28-day study results of Oleyl-diamine dioleate form an excellent match with the results from the 28-day on Oleyl-diamine. This makes clear that the toxicological profile of Oleyl-diamine dioleate is fully determined by its Oleyl diamine content. The 28-day study on Oleyl-diamine resulted to a NOAEL 1.25 mg/kg bw/day.

 

C12-14-alkyl-diamine:

- 28-dayoral gavage rat

The evaluation of the repeat toxicity of C12-14-diamine is based on a 28 day and 90-day study performed according to OECD guideline and under GLP:

 

The 28 -day study was performed at dose levels of 0, 0.4, 1.5 and 6 mg/kg bw/day administered per gavage for 28 days. An additional group of animals were subjected to the same treatment but with a 14-day recovery. Relatively minor changes in haematological, i.e. increase in neutrophils and decrease in lymphocytes, and clinical chemistry (total protein) parameters were observed in the high dose group. The finding of the most significant toxicological relevance is the histopathological changes in small intestine and mesenteric lymph nodes which indicated accumulation of foam cells. These changes regressed, but were still present, following the 14-day recovery period. A NOAEL of 0.4 mg/kg bw/day was established based on these findings.

 

- 90-dayoral gavage rat

The 90-day study was performed 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 are attributed to gavaging 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, such as liver, kidney, adrenal glands and 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, i.e. the ileum and jejunum, mesenteric lymph nodes, 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 show regression during the recovery period, but were still present after 90 days.

Evaluation:

The most significant treatment-related changes in all studies are increased WBC (neutrophils) at higher dose levels combined with changes in body weight gain. The critical effects are histopathologically, test item-relatedeffects on the small intestine and mesenteric lymph nodes, which only partially regress during the recovery period. A relatively strong inflammatory reaction is also observed. These effects have consistently been observed with these sort of substances (Fatty amine derived substances), which support the current approach of grouping of substances and read-across of data. When taking into consideration the relatively strong 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. A mode of action has not been established but it is possible to suspect the known corrosivity to be at least partially involved.

The observed effects of foamy macrophages are local and are by some interpreted as phospholipidosis (PLDsis), something which is commonly observed following treatment with cationic amphiphiolic drugs and considered to be non-adverse.

 

When comparing the 28-day results on Oleyl-diamine dioleate with the 28-day study on Oleyl diamine clearly shows a comparable toxicological profile.For both studies the effects observed, as well as the levels of the effects are very similar resulting to the same NOAEL when based on Oleyl-diamine content: 1.8 mg Oleyl-diamine/kg bw for Oleyl-diamine dioleate (based on NOAEL of 5 mg/kg bw/day for Oleyl-diamine dioleate itself) and 1.25 for Oleyl-diamine (differences are related to dose selection per study rather than actual differences in response).

Also, the further studies on C12-14-diamine show the same toxicological responses.

The results from C12-14-diamine are considered to represent a worst-case situation for Oleyl-diamine (and thus also for Oleyl-diamine dioleate), as both substances share the same molecular structure, but due to its overall smaller alkyl chain length, its solubility and potential to pass cell-membranes is expected to be possibly somewhat higher than that of Oleyl-diamine. Therefore, any inherent hazards for systemic toxicity are more likely to be expressed when testing with C12-14-diamine than with Oleyl-diamine.

 

All findings observed in the 90-day repeated dose toxicity study are comparable with the 28-day repeated dose toxicity study performed with C12-14-diamine, which indicates that duration has no great impact on the NOEAL.

Small intestinal and mesenteric lymph node lesions show only partial and slowly reversibility. The effects were not reversible during the 28-day recovery period, but had partially regressed after the 90-day recovery period. Other effects seen at higher dose levels as spleen, bone marrow and tracheal changes had completely regressed after the 28-day and 90-day recovery period.

In conclusion:

The above data shows that 28-day studies on Oleyl-diamine dioleate and Oleyl-diamine show the same toxicological profile and NOAEL when based on Oleyl-diamine content. This also indicates the possibility of cross-reading from alkyl-diamines to the alkyl-diamine mono-oleates. The data on C12-14-diamine can be taken as representative for the alkyl-diamines, and the 90-day NOAEL can be taken considered a worst-case approach for the alkyl-diamines with higher alkyl chain-lengths than C12-14.

Taking the 90-day NOAEL of 0.4 mg/kg bw for C12-14-diamine as NOAEL for Oleyl-diamine (as worst case), this would equal to a NOAEL of 0.75 mg/kg bw on the basis of Oleyl-diamine mono-oleate salt. This value is taken for the starting point for DNEL calculations for Oleyl-diamine mono-oleate (mw oleyl-diamine = 324.7, mw oleyl-diamine mono-oleate = 607.1; NOAEL 0.4 mg/kg bw x 607.1/324.7 = 0.75 mg/kg bw).

 

An additional observation is that the NOAEL for Oleyl-diamine dioleate based on 90-day (sub-chronic) study on C12-14-diamine (NOAEL 1.1 mg/kg calculated as Oleyl-diamine dioleate) is lower than the NOAEL from the 28-day (sub-acute) study on Oleyl-diamine dioleate itself (NOAEL 5 mg/kg bw) when corrected for the assessment factor of 3 for sub-acute to sub-chronic extrapolation: 5/3 = 1.67 mg/kg bw. The data indicates that duration of the study is of nogreat impact to the NOAEAL, and this lower value can therefore be considered as an extra level of conservatism in the risk assessment.

 

DERMAL

Manufacture and use are highly controlled. Its use is limited to industrial and professional users where its corrosive properties will provide for sufficient protection measures to prevent exposure. Furthermore, dermal uptake can be expected to be very limited, and as consequence, effects will be characterised by local irritation rather than by systemic toxicity following dermal uptake.The skin is therefore not a preferred route for testing to evaluate systemic toxicity following repeated dose.

 

INHALATION

Likelihood of exposures via inhalation is low considering thatOleyl-diamine mono-oleate is a paste with a vapour pressure of ≤ 0.00087 Pa at 25 °C. Its use is limited to industrial and professional users and does not involve the forming of aerosols, particles or droplets of an inhalable size. So, exposure to humans via the inhalation route will be unlikely to occur. Furthermore, considering the low vp, inhalation study would involve exposure via aerosols, and with the substance being classified as corrosive, the effects by inhalation will rather be characterised by local irritation than systemic toxicity.

Justification for classification or non-classification

The available data results to a classification to STOT-RE Cat.2 with H373: May cause damage to organs(morbidity or death)through prolonged or repeated exposure

 

STOT-RE Cat,1 is required in case of significant toxicity at levels at levels ≤ 10 mg/kgbw/d in 90-day studies or ≤ 30 mg/kgbw/d in case of 28-day studies. For Cat.2 classification levels up to 10 times higher apply.

Evaluation for classification is based on:

- read-across data from Oleyl diamine and Oleyl-diamine dioleate showing that hazard is fully related to the Oleyl-diamine content and is not affected by the oleic acid content.The data on Oleyl-diamine dioleate fully agrees to cross-reading to diamines (Fraction of oleyl-diamine is 36.5%). Consequently cross-reading to the full repeated dose endpoint data of Oleyl-diamines can be applied for Oleyl-diamine mono-oleate:

- read-across from the available data C12/14-alkyl-diamine. Only for the C12/14-alkyl-diamine a 90-day study is available which can be considered to lead to a conservative conclusion for Oleyl diamine, and subsequent for Oleyl-diamine mono-oleate.

 

The available data on Oleyl-diamine and C12/14-diamine indicate that all diamines based on alkyl chain lengths between C12/14 and C18 (represented by Oleyl) should be classified Cat.1 for STOT-RE.

- 90-day study with N-C12/14 alkyl-1, 3-diaminopropane: This study showed significant toxicity at the highest tested dose of 6 mg/kgbw/day based on mortality observed in the highest dose group. This level would be equivalent to the amount of Oleyl-diamine in about a little over 11 mg/kg bw/day of Oleyl-diamine mono-oleate. As this is above 10 mg/kg bw, but below 100 mg/kg bw it therefore results to classification STOT-RE Cat.2.

- 28-day study with Oleyl-diamine: This resulted to significant toxicity (mortality) at the highest dose of 20 mg/kgbw/day with effects on weight of body and organs without histological changes. This level would be equivalent to the same the amount of Oleyl-diamine in about a little over 37 mg/kg bw/day of Oleyl-diamine mono-oleate. This is above 30 mg/kg bw, but below 300 and therefore also results to classification STOT-RE Cat.2.

- 28-day study with Oleyl-diamine dioleate:The available 28-day study on Oleyl-diamine dioleate resulted to a NOAEL of 5 mg/kgbw. The highest tested dose of 100 mg/kg results possibly not to sufficient for severe toxicity, although indications of disseminated inflammatory response with increased WBC all animals and myeloid hyperplasia in the sternal bone marrow in 3/5 males were reported. A range finding study with 400 mg/kg was too toxic within 10 days (400 mg is equivalent to 272 mg Oleyl-diamine mono-oleate on the basis of same levels of Oleyl-diamine). These data also indicate classification STOT-RE 2.

 

Comment: Granulomas can arise from a variety of sources and syndromes, and may form in many parts of the body. The process involves macrophages and the body’s response to a “foreign” substance (often a microbe). Lipids are one type of substance that is known to induce this type of immune system response. Possibly that necrotizing granulomas can result from leakage of the natural toxic substances (proteases) released from macrophage lysosomes. Even inert substances, such as sutures, have been reported to cause granulomas in the body, sometimes well after insertion, and that most caseating (necrotic) granulomas most commonly occur in tuberculosis. Some texts refer to chronic inflammation of lymph nodes resulting in granuloma formation as “injury” to the lymph node and the GHS guidance for STOT-RE does speak about granuloma formation in vital organs as an example of a relevant toxic effect in humans.