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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to the read across-justification provided in IUCLID section 13.
Cross-reference
Reason / purpose for cross-reference:
read-across source

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Propane-1,3-diol
EC Number:
207-997-3
EC Name:
Propane-1,3-diol
Cas Number:
504-63-2
Molecular formula:
C3H8O2
IUPAC Name:
propane-1,3-diol
Specific details on test material used for the study:
1,3-propanediol (99 % pure) was provided by Shell Chemical Company, Houston, TX

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: 160-201 g for mlaes; 138-158 g for females
- Fasting period before study: None
- Housing: Individually in wire-mesh cages suspended over cage board
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72±4°F
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on oral exposure:
Crl:CD (SD) BR rats (10/sex/group) were administered 1,3-propanediol by oral gavage daily for 91 or 92 days at concentrations of 100, 300 and 1000 mg/kg/day. A concurrent control group (10/sex) was administered the vehicle, deionized water. The dose volume for all groups was 10 mL/kg.

PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test substance was weighed into a precalibrated, labeled container and deionized water added to the calibration mark. The preparations were continuously throughout the dispensation and dosing procedures using magnetic stir bars and plates. The dosing solutions, including the vehicle, were prepared weekly and were stored refrigerated until dispensation.

Individual doses were adjusted weekly based on the most recent body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the study, 10-mL samples were collected from the middle stratum of the low- and high-dose formulations, and stability was determined at 0, 1, 8, and 15 days. During the study samples for concentration verification were collected from the middle of each dosing formulation for the first 4 weeks of dosing and monthly thereafter.

Samples were analyzed via gas chromatography with FID (flame ionization detection).

One and 10% concentrations of 1,3-propanediol in water were stable for up to 15 days. Dosing solutions were generally within 20% of nominal at the 10 mg/mL concentration and within 4% at the 30 and 100 mg/mL concentrations.
Duration of treatment / exposure:
91-92 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Doses were selected based on the results of a 14-day gavage study in rats in which no adverse effects were seen at doses up to 1000 mg/kg bw/day.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed daily at the time of dosing and 1-2 hours post-dose. Detailed clinical observations were performed weekly and prior to euthanization.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: Ophthalmologic examinations were conducted prior to study initiation and termination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 and at scheduled necropsy
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10/group
- Parameters checked: The following hematological parameters were measured: total leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, prothrombin time, activated partial prothrombin time and the differential leukocyte count (% and absolute) of neutrophils, lymphocytes, monocytes, eosinophils and basophils.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 and at scheduled necropsy
- Animals fasted: Yes
- How many animals: 10/group
- Parameters checked: The following serum chemistry parameters were measured: albumin, total protein, globulin, albumin/globulin ratio, total bilirubin, urea nitrogen, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, glucose, total cholesterol, calcium, chloride, phosphorus, potassium and sodium.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

Complete necropsies were performed on all animals that survived to scheduled termination. The following organs were weighed: adrenals, brain, epididymides, kidneys, liver, ovaries (with oviducts), pituitary, prostate, seminal vesicles with coagulating glands, testes and thyroid.

HISTOPATHOLOGY: Yes

The following tissues and organs were collected and preserved appropriately: adrenals, aorta, bone with marrow (sternebrae), bone marrow smear from femur, brain (forebrain, midbrain and hindbrain), coagulating gland, epididymis (right), eyes with optic nerve, gastrointestinal tract (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon and rectum), heart, kidneys, liver (sections of 2 lobes), lungs (with bronchi and fixed), mesenteric and submandibular lymph nodes, mammary glands (females only), ovaries with oviducts, pancreas, peripheral nerve (sciatic), pituitary, prostate, submaxillary salivary glands, seminal vesicles, skeletal muscle (vastus medialis), skin, spinal cord (cervical, midthoracic, lumbar), spleen, testis (right), thymus, thyroid (with parathyroids, if present), trachea, urinary bladder, uterus with vagina, vas deferens and all gross lesions. After fixation, these tissues were trimmed, sectioned and examined microscopically for all of the animals in the control and high dose groups. Also, the lungs, liver, kidneys, stomach and testes were examined microscopically from all animals in every group.
Other examinations:
Spermatogenic endpoints were evaluated for all males at study termination. See Reproduction Section (7.8) for details.
Statistics:
All data analyses were conducted using two-tailed tests, p < 0.01 and p < 0.05, comparing the treated groups to the control group by sex. Standard deviations were calculated for all means. Body weight, body weight change, food consumption, clinical pathology, absolute and relative organ weight data were subjected to a one-way analysis of variance, followed by Dunnett's test. Clinical laboratory values for leukocytes that occur at a low incidence (monocytes, eosinophils and basophils) were not subjected to statistical analysis. In addition, the statistical analysis was not performed if the number of animals was 2 or less. All statistical tests were performed by a computer with appropriate programming.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and body weight gains were unaffected at all dose levels. A statistically significant increase in mean body weight gain for the 300 mg/kg/day group males during Week 6-7 was attributed to the substantial body weight gain of 42 g for a single animal.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There were several statistically significant differences noted in white blood cell parameters in the treatment groups as compared to the control group. See Table 1. Mean white blood cell and/or lymphocyte counts for all treated males were decreased at the Week 4 interval. However, the differences were attributed to high control group values and not test article administration. At the Week 13 interval, the mean absolute lymphocyte value was increased in the females treated at 1000 mg/kg/day; however, this value was similar to the value for this group at the Week 4 interval and a similar increase was not seen in the males in this treatment group. As no relationship to treatment was established for these differences, no treatment-related hematological changes were observed for any of the treatment groups at either interval.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Comparison of collected serum chemistry values at both intervals identified some statistically significant differences for the female treatment groups as compared to the control group. See Table 2. At Week 4, decreases were observed in mean aspartate aminotransferase (AAT) at 300 and 1000 mg/kg/day (statistically significant only in females), in mean cholesterol in females at 100 mg/kg/day and in mean chloride at 1000 mg/kg/day in females. Mean glucose was increased at all dose levels but statistically significant in females only. In general these parameters are highly variable and all differences from the control group were slight and/or not present in a dose-related manner. At Week 13, total bilirubin was decreased in females at 100 and 1000 mg/kg/day; however, slight decreases in bilirubin are not usually considered toxicologically significant. There were no other notable differences in the serum chemistry parameters examined; and none of the noted differences are considered to be treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Findings in the treated groups were limited to one or two animals in various groups, occurred similarly in the control group and/or were findings commonly observed in laboratory rats.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Findings in the treated groups were limited to one or two animals in various groups, occurred similarly in the control group and/or were findings commonly observed in laboratory rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Spermatogenic endpoints were evaluated for all males at study termination. No treatment-related effects on spermatogenic endpoints were observed at any dose level. See Section 7.8.1 for results.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted at the highest dose level tested
Dose descriptor:
LOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted at the highest dose level tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Hematology parameters























































Hematology Parameter



Males (mg/kg/day)



Females (mg/kg/day)



 



0



100



300



1000



0



100



300



1000



Week 4


White Blood Cells (thous/µL)



18.2



14.1*



13.3^



14.4*



10.0



9.3



8.9



11.9



Week 4 Lymphocyte Count (thous/uL)



14.4



11.0*



10.2^



11.3



7.5



7.3



6.9



9.6



Week 13 Lymphocyte Count (thous/uL)



11.0



9.8



9.9



10.7



7.7



9.4



7.0



9.6*



* p< 0.05; ^ p< 0.01


Table 2: serum chemistry parameters













































































Serum Chemistry Parameter



Males (mg/kg/day)



Females (mg/kg/day)



 



0



100



300



1000



0



100



300



1000



Week 4


AAT (U/L)



104



91



87



98



113



98



90^



87^



Week 4 Cholesterol (mg/dL)



53



53



48



47



68



52^



63



63



Week 4 Chloride (mEq/L)



101



101



100



100



103



103



103



100^



Week 4 Glucose (mg/dL)



115



120



121



121



103



117*



119*



118*



Week 13 Total Biliruben (mg/dL)



0.1



0.1



0.1



0.1



0.3



0.2*



0.2



0.2*



* p< 0.05; ^ p< 0.01

Applicant's summary and conclusion

Conclusions:
NOAEL: 1000 mg/kg (highest dose tested)
Executive summary:

There are no experimental data on mammalian toxicity available with the test item.


However, comprehensive data from the source substance Propane-1,3-diol is taken into account for assessment of the toxicological properties of the registered substance together with the assessment of the second source substance Octanoic acid (taken from the disseminated ECHA REACH-Dossier of Octanoic acid), according to REACH Annex XI, 1.5 (Read-Across). The target substance is the ester of the two source substances. For the respective read-across justification please refer to IUCLID Section 13.


Read across to Propane-1,3-diol


For the source substance Propane-1,3-diol a publication is available describing a subchronic toxicity study with rats (oral administration). The study followed OECD test guideline 408 and was conducted in compliance with GLP. The NOAEL of orally administered propane1,3-diol was determined to be 1000 mg/kg/day (the highest dose tested).


Read across to Octanoic acid


The second source substance, Octanoic acid, was assessed for its oral repeated dose toxicity by means of a read across approach applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. Data obtained from oral repeated dose toxicity studies with members of the category of glycol esters were therefore analysed. The Glycol ester category covers esters of an aliphatic diol (ethylene glycol, propylene glycol or 1,3-butyleneglycol) and one or two carboxylic fatty acid chains. The fatty acid chains comprise carbon chain lengths ranging from C6 to C18, mainly saturated but also mono unsaturated C16 and C18, branched C18 and epoxidized C18. The following results are obtained from the disseminated ECHA REACH Dossier of Octanoic acid:


Subchronic (84 days, rat): NOAEL oral ≥ 12500 mg/kg bw/day; CAS# 112-80-1, C18:1


Subacute (OECD 422, rat): NOAEL oral ≥ 1000 mg/kg bw/day; CAS# 112-85-6, C22


Subacute (OECD 407, rat): NOAEL oral ≥ 1000 mg/kg bw/day; CAS# 112-05-0, C9


Subacute (OECD 407, rat): NOAEL oral ≥ 1000 mg/kg bw/day; CAS# 70321-72-1, C8-18 and C18-unsatd, distn. residues


In conclusion, no human hazard for systemic toxicity after repeated oral exposure was identified for the members of the category.


As stated in the disseminated Dossier of Octanoic acid, fatty acids are found in all living organism fulfilling three fundamental roles. Besides their function as part of molecules like phospholipids and glycolipids important for the cell-structure, they are often precursors of signalling molecules such as prostanoids. The third and best understood role of fatty acid is their role as nutritional energy source. Based on their physiological function within the body no toxicity after repeated administration of fatty acids is expected as demonstrated by animal studies with C6 fatty acid (hexanoic acid), C9 fatty acids (nonanoic acid and azelaic acid), C12 fatty acid (lauric acid), C22 fatty acid (docosanoic acid), C18:1 fatty acid (oleic acid), C18:2 conj. fatty acids (conjugated linoleic acid), C18:3 fatty acid (linolenic acid), fatty acids, C8-18 and C18-unsatd, distn. residues and fatty acids, tall oil.


In the disseminated Dossier of Octanoic acid the following summaries of the study data can be found:


Subchronic


A subchronic oral toxicity study was performed with oleic acid (CAS# 112-80-1) in rats (1969). The animals (10/sex/dose) were fed a diet containing 5, 10 and 25% oleic acid equivalent to dose levels of 2500, 5000 and 12500 mg/kg bw/day for 84 days. A control group was administered the plain diet. Two control animals and one female animal from the 10% dosage group died from blood collection trauma. There were no other mortalities or clinical signs of toxicity and no abnormal behavioural reactions were noted. The test animals had slightly higher final body weights than controls, but the differences were not statistically significant. Food consumption among test animals was slightly lower than among the control animals. There were no outstanding differences in haematologic, clinical chemistry or urinalysis parameters among test and control animals. No abnormalities were noted at gross examination. There were no significant differences in organ/body weight ratios except for kidneys, adrenal glands and brain. For these three organs, female test animals showed a higher organ/body weight ratio than control animals. All of these differences could be attributed to the slightly higher body weights observed in every test group rather than a deleterious effect of the test material. The absence of any abnormalities of these organs upon histopathologic examination would support this conclusion. Some minor histopathologic changes were noted among both the test and control animals, specifically involving lesions in the trachea and lungs. These changes were judged to be due to spontaneous disease (potential pneumonia), and not related to the test material. Based on these results, a NOAEL of ≥25% oleic acid in diet was derived, corresponding to a dose level of 12500 mg/kg bw/day.


 


Subacute


Nonanoic acid (CAS# 112-05-0) was tested in a subacute 28-day study according to OECD guideline 407 and in compliance with GLP (2002). Wistar rats (5 rats/sex/dose) were administered by gavage dose levels of 50, 150, 1000 mg/kg bw/day at a frequency of 7 doses per week. No substance-related mortality occurred. Slight to moderate breathing difficulties (rales and/or gasping) were observed in several high dose animals on some days during week 3. These signs subsided and were absent during week 4. Thus, these clinical signs (generally minimal to mild) were regarded as being of no biological relevance. No signs were noted in the low and intermediate dose groups. Body weights and body weight gain were similar across controls and treated groups. There was no effect on haematology and clinical chemistry parameters. No changes were seen in hearing ability, pupillary reflex, static righting reflex and grip strength, or in motor activity in neurobehavioural functional tests.


At termination, absolute and relative organ weights were similar between control and treated animals. An irregular surface of the forestomach was noted in all high dose animals at necropsy. A thickened glandular mucosa of the stomach was observed in animals receiving the test substance. Since none of these cases could be confirmed microscopically, they were considered to be of no toxicological relevance. Histopathology revealed no other findings than slight to marked hyperplasia of the squamous epithelium of the forestomach in all high dose animals, and at a minimal degree in 3 animals of the 150 mg/kg bw/day dose group.


Based on these results, and taking into account that there is no correlate for the rat’s forestomach in humans the NOAEL for systemic toxicity is considered to be ≥1000 mg/kg bw/day. The effects noted in the forestomach are considered to reflect local irritation at the point of contact. Therefore, the NOAEL for local toxicity is considered to be 150 mg/kg bw/day.


The repeated dose toxicity of docosanoic acid (CAS# 112-85-6) was evaluated in a combined repeated dose and reproductive/developmental toxicity screening test performed under GLP according to OECD guideline 422 (2002). Groups of 13 male and 13 female Sprague-Dawley rats received daily doses of 100, 300 and 1000 mg/kg bw/day of docosanoic acid by gavage, respectively. While the males were treated for 42 days, the females received the test substance from 14 days prior to mating until day 3 of lactation. As a result of this treatment, neither mortality nor abnormalities in general condition were observed. In addition, no changes in body weight, body weight gain and food consumption were found. The observed minor changes in the corpuscular haemoglobin concentration, glucose, chloride, calcium and alkaline phosphate levels were regarded as incidental which also holds true for the observed changes in liver weights in male and kidney weights in females, respectively. All histopathological findings noted in all dose groups were also detected in the control groups, so that all findings could be regarded as not treatment-related. Overall, no treatment-related adverse effect was apparent, so that the highest dose of 1000 mg/kg bw/day is regarded as the NOAEL for docosanoic acid.


An oral 28-day repeated dose toxicity study was performed equivalent to OECD guideline 407 with fatty acids, C8-18 and C18-unsatd., distn. residues in male and female Wistar rats (1983). Ten animals each per sex were treated by gavage with the test substance at 50, 250 and 1000 mg/kg bw/day dissolved in olive oil. Control animals received the vehicle only. The animals were treated 5 days/week resulting in a total of 21 applications within 28 days. No mortalities or abnormal findings occurred in any dose group. No treatment-related effects on body weight gain, clinical chemistry, haematology and urinalysis were observed. No abnormal findings were seen at gross pathology and organ weight determinations. Histopathological examinations revealed inflamed edemas and ulcerations of the forestomach in the low- and mid-dose group as well as in the controls. In the highest dose group, the number of test animals with inflamed edemas and ulcerations in the forestomach were clearly reduced in comparison to the controls. In addition, the signs of local irritation were completely reversed in the recovery animals. Therefore, this effect was related to the vehicle. No treatment-related effects were observed. Therefore, the NOAEL was determined to be ≥ 1000 mg/kg bw/day.


 


Fruthermore there are several publications available, which also point out that fatty acids do not have toxic properties after repeated administration. In general, the following repeated dose studies reported in these publications were not performed according/similar to current guidelines and examine partially only less parameters:


Chronic


The effects of long-term feeding of conjugated linoleic acid (41.9% c9,t11 and 43.5% t10,c12) in Fischer 344 rats were examined by Park et al. (2005). A limited number of weanling male rats were fed either a control diet (n=10) or a diet containing 1% (corresponding to 500 mg/kg bw/day) conjugated linoleic acid over a time period of 18 months. After 12 weeks, 3 control and test animals were randomly selected, sacrificed, and subjected to body fat analysis and water content. At the end of the study period, all animals were euthanized and examined for gross pathological changes and appropriate tissues were examined histopathologically. Organ weights, clinical chemistry, and haematological parameters were determined. Survival rate, weight gain and water consumption did not differ between treatment and control groups. Food consumption was significantly lower in the group fed conjugated linoleic acid compared to controls, however since body weight of test animals did not differ to that of control animals, this effect is not considered adverse. Body fat analysis and water content at 12 weeks revealed no significant difference between groups in percentage body fat, empty carcass weights, or percentage body water. Blood glucose levels were significantly lower and mean corpuscular volume was significantly higher in animals fed conjugated linoleic acid compared to controls. As there were no difference in haematocrit, the observed differences do not indicate health concern. Blood urea nitrogen and cholesterol levels were elevated beyond the normal range in both groups, but were not significantly different between groups. Protein was detected in the urine of animals from both groups; however, the protein levels in the rats fed conjugated linoleic acid were significantly lower than that of the control group. All animals from both groups had chronic renal diseases (chronic interstitial nephritis, nephrosis, and/or glomerulosclerosis). The chronic renal failure was thought by the authors to be due to the high protein content of the diets and was not considered to be compound-related. There were no significant differences between groups in organ weights. The incidences of pituitary or testicular tumors, prostatitis, or lymphoma were not significantly different between groups. Based on the results of this study, the NOAEL was considered to be ≥ 500 mg conjugated linoleic acid/kg bw/day in the diet.


In another chronic study with conjugated linoleic acid, weanling male rats received a diet containing 1.5% of a synthetic conjugated linoleic acid preparation (42.5% c9,tll and/or t9,cl1 isomers, 43% t10,c12, 4.3% other conjugated linoleic acid isomers, 7.1% linoleic acid, and 3.1% other constituents (not further specified)) for 9 months (Scimeca, 1998). Body weights and food consumption were recorded and physical examinations were performed weekly throughout the study. At the end of the study period, all surviving animals were necropsied and examined histologically and organ weights were determined. Haematological and clinical chemistry parameters were measured. According to the authors rats ingested an average daily conjugated linoleic acid dose in a range from 1970 ± 11 to 467 ± 52 mg/kg bw/day from week 1 to study week 36. However, since this is a broad range, an average conjugated linoleic acid intake of 750 mg/kg bw/day was calculated based on an average food consumption of 5g/100g bw/day (WHO, 1987). Animals fed conjugated linoleic acid did not show any clinical signs of toxicity, nor were there any differences in body weight gain or food consumption relative to the control group.


Likewise, there were no significant compound-related histopathological or microscopic changes in the organs, or changes in haematological or clinical chemistry parameters. Based on the results of this study, the NOAEL for conjugated linoleic was determined to be 750 mg/kg bw/day.


Subchronic


Repeated dose toxicity of lauric acid was tested in a study, where 5 male Osborne-Mendel rats were fed a diet containing 10% lauric acid for 18 weeks (Fitzhugh et al., 1960). As results, no clinical effects, no adverse effects on weight gain nor any mortality were noted. The performed gross organ pathology did not reveal any significant differences of individual organ weights between the controls and test animals. Thus, the test concentration of 10% in diet is regarded as the NOAEL, which corresponds to ca. 5000 mg/kg bw/day, based on an average daily food consumption of 5 g/100 g bw/day.


Investigations on the repeated dose toxicity of azelaic acid (CAS# 123-99-9) were carried out in Wistar rats and New Zealand rabbits, fed azelaic acid incorporated into pellets (Mingrone et al., 1983). Rats (10/sex/dose) were given azelaic acid at dose levels of 140 and 280 mg/kg bw/day and rabbits (10/sex/dose) were administered azelaic acid at doses of 200 and 400 mg/kg bw/day for 180 days, respectively. Both species showed normal growth compared to the control animals. No effects on biological parameters of haematology and clinical chemistry were observed and there were no findings at histological examinations (liver, kidneys, suprarenal glands, intestine, testicles, ovaries, uterus, lung, heart and brain). Thus, the NOAELs for the subchronic toxicity study were regarded to be ≥ 280 mg/kg bw/day for rats and ≥ 400 mg/kg bw/day for rabbits, respectively.


The repeated dose toxicity of fatty acids, tall oil (CAS# 61790-12-3) which consists predominantly of C18 unsaturated and saturated fatty acids was examined in a 90-day subchronic toxicity study in Charles River rats (Pine Chemical Association, 2004). The test substance was administered to the animals (10 rats/sex/dose) in the diet at concentrations of 0, 5, 10 and 25% (corresponding to 2500, 5000 and 12500 mg/kg bw/day). No deaths occurred in the test animals; however two control animals died during blood sampling. No clinical signs and no changes in body weight and body weight gain were noted. The food consumption was slightly decreased in the mid- and high-dose group. No changes in haematology, clinical chemistry or urinalysis parameters were determined. At gross pathology, no treatment-related effects were observed in any of the groups. No consistent organ weight changes and no histopathological effects were reported at any dose. Based on this data, the NOAEL for fatty acids, tall oil was considered to be ≥ 12500 mg/kg bw/day.


Subacute


Moody and Reddy (1977) exposed rats to 2, 4 and 8% hexanoic acid (corresponding to 1000, 2000, 4000 mg/kg bw/day; CAS# 142-62-1) in diet for 3 weeks before alterations in body weight gain, liver size, hepatic enzyme activity and hepatic peroxisome proliferation were examined. Since no effects were induced by hexanoic acid, the NOAEL was considered to be ≥ 4000 mg/kg bw/day.


Rodrigues et al. (2010) analysed effects of oleic acid (CAS# 112-80-1) and linoleic acid (CAS# 60-33-3) after repeated dose exposures in male Wistar rats with special focus on neutrophils. In detail, 10 rats per dose group were exposed once daily to 110, 220 and 440 mg/kg bw oleic acid or linoleic acid via gavage over 10 days. Control animals received 220 mg water/kg bw /day. In addition to specific alterations in neutrophils, effects on water, food and calory intake as well as alterations in biochemical parameters including enzyme activities of alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) were determined Moreover histological examinations on the small intestine including villus:crypt ratio, epithelium, reactivity of the crypt, claciform cells number, payer plates reactivity, muscle layer and nerve plexus were performed. In regard to signs of general toxicity, no mortality or clinical signs such as diarrhoea or hair loss were reported. Food, calorie and water intake were not modified by administration of oleic acid and linoleic acid in all dose groups. Moreover, no alterations in the activity of AST, ALT or LDH were observed in the high-dose group and no changes in the histopathological examinations were determined. Oleic acid and linoleic acid modified several neutrophil functions, indicating that these fatty acids may affect the course of inflammation. As the effects on neutrophils represent cell-specific alterations relevant for the immune system which itself represents an adaptive system to many kinds of stressors and exogenous stimuli, they do not clearly indicate adverse effects on the test animals. In contrast, endpoints covering clinical signs, mortality, food and water consumption, haematological parameter and histopathological findings clearly stand for no significant adverse effects of oleic and linoleic acid. Thus, the highest dose of 440 mg/kg bw is regarded as NOAEL in the conducted study for both test substances.


The effect of linoleic acid on the haemopoietic system was investigated in rats (Khan et al., 1994). Five animals each were administered linoleic acid (CAS# 60-33-3) in mineral oil at a dose level of 0.7 mmol/kg bw/day (equivalent to 200 mg/kg bw/day) by gavage for 1, 7 or 28 days, respectively. No haematological changes were noted in the examined parameters (blood erythrocyte count, haemoglobin, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, haematocrit, platelets, white blood cell count, leukocyte differential counts and methaemoglobin). Biochemical assays in serum revealed, that LDH activity was not altered by linoleic acid treatment. Serum AST was diminished to 72% and 74% of control values in animals treated for 7 and 28 days, respectively. Serum ALT activity was decreased in the linoleic acid exposed animals at Days 1 and 7 being 81 and 65% of controls, respectively. On day 28 the changes in serum ALT activity were no longer of statistical significance. Since the changes in these markers of cell or tissue injury were not accompanied by any histopathological findings in the examined organs (heart, liver, lung, brain, spleen, kidneys, thymus, testes and pancreas), the changes in the aminotransferases were not considered as adverse. No organ weight changes were noted and linoleic acid treatment had no effect on splenic iron content at any of the three time points. Thus, based on the results of this study, the NOAEL (subacute) was considered to be ≥ 200 mg linoleic acid/kg bw/day.


Since the members of the fatty acids category share structural and functional properties, these study results can be applied to all members of the category. Thereby, a substance-specific adjustment of the NOAEL is not performed. As overall NOAEL for all fatty acids within the category 1000 mg/kg bw/day is chosen as “worst case” among the available key studies, which were judged with reliability 1 or 2 (reliable). In addition nonanoic acid and docosanoic acid, which elicit a NOAEL ≥ 1000 mg/kg bw/day are representing category members with a low and the highest molecular weight.


It is concluded in the disseminated Dossier of Octanoic acid that the study data do not provide any evidence of systemic toxicity after repeated administration of fatty acids which is supported by the physiological function of fatty acids within the body.


 


References


Fitzhugh, O.G. et al. (1960) Oral toxicities of lauric acid and lauric acid derivates. Toxicol Appl Pharmacol. 2:59 - 67.


Khan, M. F. et al. (1994) Hematopoietic Toxicity of Linoleic Acid Anilide: Importance of Aniline. Fundam Appl Toxicol 25:224 - 232.


Mingrone, G. et al. (1983). TOXICITY OF AZELAIC ACID. DRUGS EXPTL. CLIN. RES. 9(6):447 – 455


Moody, D. E. and Reddy, J.K. (1978). Hepatic Peroxisome (Microbody) Proliferation in Rats Fed Plasticizers and Related Compounds. Toxicol. Appl. Pharmacol. 45:497 – 504.


Park, Y. et al. (2005).Effects of conjugated linoleic acid on long term feeding in Fischer 344 rats. Food and Chemical Toxicology 43:1273 – 79.


Pine Chemical Association (2004). Final Submission for Tall Oil Fatty Acids and Related Substances - VII. Robust Summaries of Data for Tall Oil Fatty Acids and Related Substances.Bibliographic source: no data


Scimeca, J.A. (1998) Toxicological Evaluation of Dietary Conjugated Linoleic Acid in Male Fischer 344 Rats. Food and Chemical Toxicology 36(5):391 - 395.


Rodrigues, H.G. et al. (2010). Dietary Free Oleic and Linoleic Acid Enhances Neutrophil Function and Modulates the Inflammatory Response in Rats. Lipids 45:809 – 819.


 


Prenatal developmental toxicity data


Besides the above mentioned data on repeated dose toxicity, data obtained from prenatal developmental toxicity studies are additionally used in this weight-of-evidence-approach to assess the repeated dose toxicity of the substance.


Two prenatal developmental toxicity studies are available with further read across substances. The source substances (CAS 68583-51-7 and CAS 91031-31-1) are UVCB substances (fatty acids with alcohols) but structurally similar to the above mentioned read across substances. For details, please refer to the read across justification. In both studies no adverse effects could be obtained up to the highest dose tested (i. e. 900 and 1000 mg/kg bw/d, respectively). Therefore, the NOAEL was determined to be the limit dose which is commonly applied for repeated dose toxicity, i. e. 1000 mg/kg bw/d. The treatment period in the pre-natal developmental toxicity study is shorter when compared to a subacute toxicity study (14 days vs. 28 days). However it should be taken into account that pregnant animals were administered being rather more susceptible to xenobiotics. Since no adverse effects were observed at all, it is considered very unlikely that a 14 days longer treatment period would lead to substantial adverse effects.


 


Use profile


In addition to the hazard assessment the use profile of the registered substance should be taken in to account. The registered substance is only handled in controlled industrial settings making considerable exposure to humans unlikely. Assessment of toxicokinetic behaviour being based on physicochemical properties of the registered substance and toxicity data of the structural analogue substances concluded that the test items bioavailability will probably be very low, if not negligible. The registered substance is also applied in cosmetic products. Here, human health safety assessment of the final product is ensured under Cosmetics Regulation 1223/2009.


 


Conclusion


Based on the available data with the read-across source substances, the target substance is considered to be of very low toxicity since no adverse systemic effects were obtained in any of the toxicity studies conducted. The read-across substances Propane-1,3-diol and Octanoic acid taken into account for the assessment of the repeated-dose toxicity of the target substance only require classification for local effects (low potential for skin and eye irritation and skin corrosion 1C, respectively) according to Regulation (EC) No 1272/2008 (CLP).


A QSAR Toolbox query (2020) with the target substance supports the above considerations. There were no structural alerts in regards to specific target organ toxicity, no categorization according to HESS and the Cramer class was predicted to be low (Class I).


Therefore, a risk assessment does not need to be conducted according to REACH Article 14(4) and DNEL derivation is not required.       


In conclusion and taking into account its low to negligible toxicity, the registered substance raises no concerns in regards to human health risk assessment that would justify a vertebrate repeated dose toxicity study. Neither as a hazard nor in regards to exposure considerations. Thus, conducting a repeated dose toxicity study in vertebrates is not justified. This is in line with REACH Article 25 (1) claiming that animal testing should always be the last resort.


 

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