Naphthenic acids

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-03 to 2018-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Naphthenic acids

EC Number:

215-662-8

EC Name:

Naphthenic acids

Cas Number:

1338-24-5

Molecular formula:

For the acidic (naphthenic) fraction: CnH2n+zO2, where n = carbon number and z = homologous group series number: z = 0 when no ring structures are present, z = -2 when 1 ring is present, z = -4 when 2 rings are present etc. For the non-acidic fraction: not applicable

IUPAC Name:

11-(decahydronaphthalen-2-yl)undecanoic acid; 11-cyclopentylundecanoic acid; 15-cyclohexylpentadecanoic acid; 3-(tetradecahydroanthracen-2-yl)propanoic acid; 4-(tetradecahydroanthracen-2-yl)butanoic acid; 7-(tetradecahydroanthracen-2-yl)heptanoic acid; 9-(octahydropentalen-2-yl)nonanoic acid; dodecahydro-1H-phenalene-1-carboxylic acid

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Umicore, 2017787NA12
- Expiration date of the lot/batch: 31 July 2019
- Purity: considered as 100 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤70 Relative Humidity%)
- Stability under test conditions: The test item was incorporated into the animal feed. The stability of the test item in the diet has been verified for a period of at least 11 weeks at room temperature.
-Solubility and stability of the test substance in the solvent/vehicle: Analysis of the diets for homogeneity and concentration of the test item was performed by an analytical method validated at the Test Site using GC-FID (gas chromatography-flame ionization detection) method to determine the Naphthenic acids content on two occasions from each batch used in the study (once at the start of feeding and once during the last week of the treatment).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was incorporated into ssniff® SM R/M-Z+H “Complete Feed for Rats and Mice-maintenance” by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, to generate the test concentrations required. The pelleting process is considered not to induce an "unmixing" of the diets or particle segregation, and would prevent the potential settling out of the fine-heavy particles that could occur in handling/transport of powder diets. Naphthenic acids was incorporated into the diet and mixed for up to approximately 16 minutes (approximately 8 minutes for premix preparation, and approximately 8 minutes for preparation of the complete diets; minor variations were acceptable as practical). Following mixing, pellets were prepared by simple compression; no binding agents, steam, external heat, any other process or substance were used that might affect the test item or the quality of the diets. Similar diet preparation procedures were used to generate control diet (0 mg Naphthenic acids/kg diet). The prepared diets were stored at room temperature, in sewed bags pending and during transport to Citoxlab Hungary Ltd. At Citoxlab Hungary Ltd., the prepared diets were stored also at controlled room temperature.

FORM AS APPLIED IN THE TEST (if different from that of starting material): The test item was incorporated into ssniff® SM R/M-Z+H “Complete Feed for Rats and Mice-maintenance”

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected as it is a readily available rodent species, historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities. Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks at start of treatment
- Weight at study initiation: Males: 254 – 297 g; Females: 181 – 221 g
- Fasting period before study: No
- Housing:
Cage type: Type II and/or III polycarbonate
Bedding: Lignocel® (produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany) Bedding for Laboratory Animals and nest building material (Arbocell crinklets natural (produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany)) were available to animals during the study
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M-Z+H “Complete Diet for Rats and Mice – Breeding and Maintenance” produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, with or without Naphthenic acids (treated or control groups, respectively), ad libitum.
- Water (e.g. ad libitum): Tap water was supplied from 500 mL bottles, ad libitum.
- Acclimation period: 14-15 days

DETAILS OF FOOD AND WATER QUALITY: Water quality control analysis was performed at least once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A. u. 36., Hungary).The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 – 25.9 °C
- Humidity (%): 32 – 75 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 May 2018 to 01 August 2018

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The dietary route was selected as it is the most relevant route of human exposure.

Vehicle:

other: Animal feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was shipped to ssniff® Spezialdiäten GmbH from Citoxlab Hungary Ltd. After arrival, the test item was incorporated into ssniff® SM R/M-Z+H “Complete Feed for Rats and Mice-maintenance” by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, to generate the test concentrations required.

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Naphthenic acids was incorporated into the diet and mixed for up to approximately 16 minutes (approximately 8 minutes for premix preparation, and approximately 8 minutes for preparation of the complete diets; minor variations were acceptable as practical). Following mixing, pellets were prepared by simple compression; no binding agents, steam, external heat, any other process or substance were used that might affect the test item or the quality of the diets. Similar diet preparation procedures were used to generate control diet (0 mg Naphthenic acids/kg diet).
- Storage temperature of food: The prepared diets were stored at room temperature

VEHICLE
- Concentration in vehicle: 1500, 4200 and 12500 ppm

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the diets for homogeneity and concentration of the test item was performed by an analytical method validated at the Test Site using GC-FID (gas chromatography-flame ionization detection) method to determine the Naphthenic acids content on two occasions from each batch used in the study (once at the start of feeding and once during the last week of the treatment). Two batches from each dose level were used during the study, thus 4 sampling occasions were performed in total.

Sampling for the measurements: Five representative test-item treated diet samples of an appropriate weight were collected at each occasion during the study, from five different locations of one representative diet container from each concentration. Additionally, at one sample was similarly taken from the control diet, for analysis of Naphthenic acids concentration (i.e a total of 16 samples per occasion). Diet samples were kept at room temperature until shipment. Samples were shipped as soon as possible after collection for concentration and homogeneity measurement to the Principal Investigator of the Test Site.

The dietary samples were delivered together with a verified sample list stating the ssniff® identification number, concentration, sampling location, sample mass and sampling date.

Acceptance criteria of the concentration analysis were set according to the analytical method validation, expected to be at 100 ± 20% of the nominal concentrations. Acceptance criteria of the homogeneity was that the CV of replicates (from 5 different locations) must be less than 10%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

A constant concentration of Naphthenic acids in diet was administered to treated groups for a 90-day treatment period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg diet

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Remarks:

Low dose, 107 mg/kg

Doses / concentrations 3

Dose / conc.:

4 200 ppm

Remarks:

Mid dose, 302 mg/kg

Doses / concentrations 4

Dose / conc.:

12 500 ppm

Remarks:

High dose , 881 mg/kg

No. of animals per sex per dose:

10 males and 10 females per dose group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The concentrations of Naphthenic acids were set by the Sponsor in consultation with the Study Director based on the available data and information from previous experimental work, including the results of a 14-day dose range finding and palatability study.
- Rationale for animal assignment (if not random): The animals were assigned to their respective dose groups by randomization based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; the software PROVANTIS v.9 was used in order to verify homogeneity/variation among/within groups. Males and females were randomised separately.
- Fasting period before blood sampling for clinical biochemistry: Yes
- Other: Before the onset of the 90-day treatment period, all animals received the same control diet for 7 days during acclimation period. The first day of dosing of each animal was regarded as Day 0. All animals underwent necropsy upon completion of the 90-day treatment period (Day 89). Blood samples for Clinical pathology examination were collected prior to necropsy.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day except on those days when detailed clinical observations are made.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first treatment (on Day 0 male/female) and weekly thereafter, in the morning hours (am) and once before necropsy.
- Observations included examinations of the skin, fur, eyes, eyeballs and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (ex. hunchback posture, etc), gait, or response to handling and to environmental stimulation. Particular attention was directed to observations for tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded with a precision of 1 g at randomisation (pre-treatment period), on the first day of treatment (Day 0, prior to start of treatment), then weekly, including on Day 89, and prior to necropsy, fasted on Day 90.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was measured with a precision of 1 g before the start of treatment (Day 0), then twice weekly during the treatment period. Control diet consumption during the pre-experimental period was measured with a precision of 1 g on Day -7 (start) and on Day -1 (end). The control diet consumption between Day -1 and Day 0 was not measured.
- Animal food consumption per cage was measured at least twice weekly and the mean weekly food consumption and daily food intake per rat was calculated. Based on food consumption data, the mean dose intake of each group was calculated for reporting purposes on the basis of mg/kg body weight/day, in addition food conversion efficiency (g/g) was calculated as [weekly body weight gain (g)/weekly food consumption (g)].

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ophthalmoscopic examination was conducted in all animals before treatment (Day -1 or -2), and in the Control group and High dose group animals during Week 11/12 (Day 82/83). Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL cyclopentolate hydrochloride) into the conjunctival sac. The evaluation was performed using a WelchAllynTM ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy
- Anaesthetic used for blood collection: pentobarbital
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: RBC - Red Blood Cell (erythrocyte) count, WBC - White Blood Cell (leukocyte) count, Hgb - Haemoglobin concentration, Hct - Haematocrit (relative volume of erythrocytes), MCV -Mean Corpuscular (erythrocyte) Volume, MCH - Mean Corpuscular (erythrocyte) Haemoglobin, MCHC - Mean Corpuscular (erythrocyte) Haemoglobin Concentration, RDW - Red Cell (erythrocyte) volume, Plt - Platelet (thrombocyte) count, MPV - Mean Platelet Thrombocyte volume, RETIC % - Reticulocyte count, NE % - Neutrophil, LY % - Lymphocyte, MO % - Monocyte, BA % - Basophil, EO % - Eosinophil, LUC % - Large Unstained Cells, APTT - Activated Partial Thromboplastin Time, PT - Prothrombin Time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameter examined: Glucose - Blood sugar concentration, T-BIL - Total Bilirubin concentration, Urea - Urea concentration, Chol - Cholesterol concentration, Creat.- Creatinine concentration, Phos. - Phosphorus concentration, Na+ - Sodium concentration, K+ - Potassium concentration, Ca++ - Calcium concentration, Cl - Chloride concentration, Tot. Prot. - Total Protein concentration, Alb. - Albumin concentration, A/G - Albumin/globulin ratio, AST/GOT - Aspartate Aminotransferase activity, ALT/GPT - Alanine Aminotransferase activity, GGT - Gamma-Glutamyl transferase activity, ALKP- Alkaline Phosphatase activity, Bile acids.


URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: Urine collection was conducted over approximately 16 hours, during an overnight period of food deprivation of animals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: LEU / Leukocyte, NIT / Nitrite, pH, PRO - Protein, GLU - Glucose, UBG / Urobilinogen, BIL / Bilirubin, KET / Ketones, BLD / ERY Blood/Erythrocytes, SG / Specific Gravity, SED / Sediment, VOL / Volume, Colour/appearance.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the treatment period, during Week 11/12
- Dose groups that were examined: all animals
- Battery of functions tested: Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested.

Parameters such as, body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.

To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. This was performed 3 times for each animal on each test day. The distance between the two resulting ink spots was measured. The fore paws of the rat were painted for any possible additional measurements.

- Motor activity: was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for a 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.

- Fore/hind grip strength: conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support.

IMMUNOLOGY: Not examined

Sacrifice and pathology:

Terminal procedures: Necropsy and macroscopic examination were performed on all animals, at the end of treatment period, on Day 90 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia.

GROSS PATHOLOGY: After exsanguination the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. In addition, bone marrow smears from the femur of each animal were prepared at necropsy. The smears were fixed, then stained but not analysed.

ORGAN WEIGHT:
- The following organs were trimmed of fat and weighed in all animals: Brain, Epididymides, Heart, Kidneys, Liver, Prostate, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Uterus including cervix, Adrenal glands, Ovaries, Thyroid with parathyroid glands, Pituitary.
- Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.

HISTOPATHOLOGY:
- The following tissues and organs were collected, fixed and embedded in paraffin wax: Lungs with bronchi (Lungs of euthanized animals were infused with formalin; 3 lobes, left, right cranial, right caudal), Skeletal muscle (quadriceps), Adrenals, Lymph node (Mandibular and mesenteric), Small intestine (Duodenum, ileum and jejunum with Peyer’s patches), Animal identification (Fixation and preservation only), Ovary, Spinal cord (Transverse sections, 3 levels - cervical, thoracic and lumbar), Aorta (Aorta thoracic and abdominal), Oviduct, Spleen, Brain, Pancreas, Sternum with marrow, Epididymis, Pituitary, Stomach, Eye with the optic nerve (If applicable, optic nerves were examined histologically only if present in routine sections), Prostate, Testis, Oesophagus, Salivary gland (including mandibular, sublingual and parotid glands), Thymus, Femur with marrow, Thyroid with parathyroid gland (If applicable, parathyroids were examined histologically only if present in routine sections), Heart (Section including both ventricles and atria, septum with papillary muscle), Tongue, Kidney, Sciatic nerve, Trachea, Large intestine (Caecum, colon and rectum), Seminal vesicle with coagulating gland, Urinary bladder, Extraorbital lachrymal gland, Uterus (Horns, body and cervix), Harderian gland, Skin (subcutis with mammary gland (inguinal)), Vagina, Liver (Liver, 3 lobes, left lateral, right medial, caudate).
- Full histopathology was performed in Groups 1 (Control) and 4 (High dose).

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 software package (within the validated Provantis system). The following decision tree was applied automatically for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.

For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related clinical signs were observed during the study.

The following individual observations were not attributed to systemic effects of the test item: One Mid dose male (#3010) had tonic convulsions on Days 76 and 89. Thin fur was seen unilaterally on the cheek and/or on the neck in one Mid dose male (#3003) from Day 77 and in one High dose female (#4507) from Day 84 until the end of the observation period.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the Mid and High dose animals, statistically significantly lower bodyweight and body weight gain values were observed during the study. The day 89 body weights of the High dose were about 17% and 15% below controls in males and females respectively; the Mid dose were about 8% and 10% below control. The low body weight and body weight gain values in the Mid and High dose groups correlated with the recorded food consumption values. It was considered that the observed effects were caused by low palatability of the treated diet, the most significant lower weight gain was in the first week of feeding, when female High dose animals lost weight. The male overall body weight gain during the study was 33% lower in the High dose group and 15% lower in the Mid dose group compared to the male Control. The female overall body weight gain during the study was 45% lower in the High dose group and 30% lower in the Mid dose group compared to the female Control.

The bodyweight and body weight gain values of the Low dose did not show any test item related effect.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Lower food consumption was measured in the Mid and High dose groups during the study, reaching statistical significance at several occasions. In general, the extent of the low food consumption was greater in the beginning of the study and the effect slowly decreased towards the end of the 90-day observation period.

Slightly lower food consumption was measured also in the female Low dose group, reaching statistical significance at a few occasions, but without consequences on body weight gain. The male Low dose food consumption did not show any test item related effect.

The recorded food consumption values correlated with the observed body weight changes.

The test item dose intake values (mg/kg bw/day) were calculated from the body weight, food intake and diet concentration. The mean achieved dose levels (combined for males and females) were 107, 302 and 881 mg/kg bw/day in the Low, Mid and High dose groups, respectively.

The aim to reach a dose intake equivalent to 1000 mg/kg bw/day in the High dose groups was not fulfilled due to the low palatability of the test item; however, the body weight effect was sufficient to ensure the High dose was acceptable for a regulatory study (see summary table in section "any other information on results").

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item-related changes compared to pre-treatment were noted in the Control and High dose groups at the terminal ophthalmoscopy examination. Thus, animals of the lower dose levels were not examined at the end of the study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed in the haematology parameters. Statistically significant differences were considered to be incidental, there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were considered to not reflect an adverse effect of the test item.

Coagulation parameters: The Prothrombin Time (PTT) was statistically (p<0.01) lower in the female Mid and High dose groups, when compared to the control. All values were considered to be in the normal range and there was no clear dose-response, the statistical differences were not considered to represent an adverse effect.
See Table 2 in section 'Any additional information on results' for detailed data.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Elevated Alkaline Phosphatase activity (ALKP) was seen in the animals. The ALKP was significantly higher in the Mid dose (p<0.05) and High dose (p<0.01) animals. The male and female High dose group mean ALKP means were out of the historical ranges, while the Mid dose mean values were below the upper limit of the ranges. The low dose values were slightly (10-22%) above the control, without any statistical significance. The observed high ALKP values indicate a dose dependent effect of the test item. There were no tissues with adverse histological changes of tissue damage, although large percentage liver weight increases were observed (about 40% and 60% relative to body weight) hence it is plausible the ALKP increases may have been related to hepatic effects of the test item. The increased ALKP at the High dose was outside the historic range, and although there was no clear evidence for which tissue, it is not possible to exclude the possibility of a relatively minor adverse effect at the High dose.

In the High dose females significantly increased potassium level was recorded which correlates with the histopathological findings in the adrenals; the values in some High dose females was outside, or close to the historic control maximal values. The male mean values were unaffected by treatment. The toxicological significance of higher potassium in High dose females is uncertain.

Besides this, all other significant differences were regarded as incidental and of no toxicological significance.
See Table 3 in section 'Any additional information on results' for detailed data.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters. The significantly lower protein presence in the male High dose group and the significantly lower presence of urine crystals in the male Low dose group are not toxicologically relevant.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.

There was no adverse effect of treatment noted during the assessment of grip strength, foot splay or motor activity. A statistically significantly lower landing foot splay in the High dose females was considered as a secondary effect of a lower body weights in the group and not a toxic effect of the test item (15% lower body weight, 27% lower foot splay; all means were well within the historical control range). Neurotoxic test items cause an increased foot splay, so a reduced foot splay does not usually indicate an adverse effect. Other differences in the neurological assessment parameters were considered as incidental and not to reflect an effect of the test item.

All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals and the Control when evaluating the overall distance travelled (0-60 min, cm). The test item did not increase or decrease the normal locomotor activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The terminal bodyweights of the Mid dose and High dose animals were lower than the controls by 8-11% and 16%, respectively, due to the lower food consumption of these animals. Therefore, it is considered that the organ / bodyweight ratios are not good indicators for organ weight changes. Thus, mainly the absolute and relative to brain weights were used for evaluation of test item-related effects.

Significantly higher absolute and relative to brain weight kidney weights were seen in the male High dose groups; however, all these values were still within the historical control ranges (up to 15% above control). The females had completely normal kidney weight values. As the observed values were within the historical control range and no microscopic changes were seen at histopathology, these organ weight changes were considered as adaptive, non-adverse changes. (The significantly higher kidney / bodyweight ratio in the male Mid dose group was considered to be caused by the lower bodyweights in this group and not because of an organ weight change.)

The absolute and relative to brain weight liver weights were also statistically higher (p<0.01) in the male and female High dose groups. The High dose female values were also out of historical control range (up to 35% above control). In the Mid dose females, the liver weights were also slightly increased. The Low dose animals had normal liver weight values. The effect was confirmed at histopathology as being a non-specific adaptive change to the high work load of the liver.

Moreover, the absolute and relative to brain weight thymus weights were slightly lower in the High dose groups, reaching statistical significance only in the males (p<0.05). The observed differences correlate with the body weight changes and therefore were considered as toxicologically not relevant. No effect was seen at histopathology in the thymuses.

Besides these, all other statistically significant weight differences were considered to be incidental and not test item-related or ascribed to differences in body weight.

See Table 1 in section 'Any additional information on results' for detailed data.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related macroscopic findings were noted at necropsy.
Bilateral dilatation with clear fluid of the uterus was seen in 6 females and it was considered as normal oestrus. The unilateral dilatation of the pelvis in two males and small ovaries in one female were considered as incidental or background.
See Table 4 in section 'Any additional information on results' for detailed data.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were seen in the liver, adrenal and thyroid glands of the High dose groups.

Minimal to moderate centrilobular hepatocellular hypertrophy was observed in 9/10 male and 10/10 female High Dose rats. This finding correlates with the increased liver weights observed in the High dose groups. It is considered that the hepatocellular hypertrophy was caused by the enzyme induction in hepatocytes in response to chemical exposure and it is a non-adverse adaptive change.

Minimal follicular cell hypertrophy was observed in the thyroid of 6/10 High Dose males. This change is considered secondary to hepatic change and most unlikely a direct toxic effect of the test item. This change is not considered to reflect an adverse effect of the test item on the thyroid gland.

Minimal to slight hypertrophy of the zona glomerulosa cells in the adrenal was observed in 8/10 High dose females and in 1/10 High dose males. Zona glomerulosa is responsive to increased serum potassium levels. In the current study, statistically significant 16.5% increase in serum potassium level was observed in High Dose females. Similar changes were not observed in High Dose males. Toxicological significance of this change observed only in females is uncertain, but there was no evidence of any clear adverse effect in this organ.
See Table 5 in section 'Any additional information on results' for detailed data.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

302 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Although none of the individual findings in the High dose group were clearly adverse, taking into account the degree of liver weight increase, and the other findings observed at the High dose, altogether the effects are considered adverse.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

881 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Detailed data tables:

Table 1 Absolute and relative values for organ weights (Mean +/- SD)

Sex	Males				Females
Groups/Conc (ppm/day)	Control (0)	Low (1500)	Mid (4200)	High (12500)	Control (0)	Low (1500)	Mid (4200)	High (12500)
Terminal bodyweight (g)	561.1 +/-41.8	568.4 +/-53.8	514.1 +/-39.4	466.9 ** +/-34.2	306.1°° +/-24.1	300.1 +/-22.6	273.6 ** +/-15.0	255.8 ** +/-15.1
	Historical control data	455 – 657			Historical control data	254 – 352
Kidneys  (g)	3.062 +/-0.194	3.205 +/-0.400	3.367 +/-0.288	3.533 ** +/-0.317	1.855°° +/-0.162	1.920 +/-0.306	1.740 +/-0.130	1.718 +/-0.143
	Historical control data	2.59 – 4.22			Historical control data	1.45 – 2.29
Kidneys / Bodyweight (%)	0.547 °° +/-0.039	0.565 +/-0.067	0.656 ** +/-0.052	0.757 ** +/-0.045	0.607 +/-0.049	0.638 +/-0.068	0.636 +/-0.036	0.672 +/-0.046
	Historical control data	0.518 – 0.761			Historical control data	0.519 – 0.748
Kidneys / Brain weight (%)	142.22° +/-18.55	141.19 +/-16.76	148.19 +/-11.02	161.33 * +/-15.55	90.77 +/-6.39	91.37 +/-12.00	85.78 +/-5.64	83.44 +/-7.20
	Historical control data	117.50 – 189.91			Historical control data	71.43 – 109.05
Liver (g)	15.052° +/-1.321	15.538 +/-1.505	15.759 +/-1.768	17.392** +/-1.554	8.661°° +/-1.033	9.176 +/-1.272	9.701 +/-0.980	11.702** +/-1.203
	Historical control data	11.22 – 22.55			Historical control data	6.37 – 10.01
Liver / Bodyweight (%)	2.687°° +/-0.207	2.738 +/-0.184	3.061 ** +/-0.186	3.723** +/-0.129	2.827°° +/-0.218	3.056 +/-0.322	3.544** +/-0.286	4.575** +/-0.375
	Historical control data	2.183 – 3.442			Historical control data	2.27 – 3.50
Liver / Brain weight (%)	697.08 °° +/-79.11	684.94 +/-62.44	693.00 +/-66.22	793.65** +/-67.81	423.23°° +/-38.43	437.12 +/-52.12	478.29 +/-45.29	568.93** +/-65.59
	Historical control data	496.28 – 939.58			Historical control data	307.73 – 489.90
Thymus  (g)	0.434°° +/-0.090	0.469 +/-0.097	0.457 +/-0.075	0.345 * +/-0.041	0.412 +/-0.067	0.397 +/-0.084	0.370 +/-0.103	0.347 +/-0.065
	Historical control data	0.26 – 0.82			Historical control data	0.18 – 0.54
Thymus / Bodyweight (%)	0.077 +/-0.014	0.083 +/-0.017	0.089 +/-0.011	0.075 +/-0.012	0.135 +/-0.021	0.132 +/-0.025	0.136 +/-0.038	0.135 +/-0.022
	Historical control data	0.052 – 0.155			Historical control data	0.067 – 0.176
Sex	Males				Females
Groups/Conc (mg/kg bw/day)	Control (0)	Low (1500)	Mid (4200)	High (12500)	Control (0)	Low (1500)	Mid (4200)	High (12500)
Thymus / Brain weight (%)	19.83°° +/-2.99	20.73 +/-4.49	20.12 +/-3.19	15.74* +/-1.88	20.08 +/-2.74	18.89 +/-3.64	18.32 +/-5.41	16.78 +/-2.63
	Historical control data	10.74 – 38.32			Historical control data	8.87 – 27.55
Brain (g)	2.176 +/-0.226	2.269 +/-0.088	2.272 +/-0.088	2.194 +/-0.115	2.044 +/-0.112	2.097 +/-0.095	2.028 +/-0.066	2.061 +/-0.091
	Historical control data	-			Historical control data	-
Brain/bodyweight (%)	0.388°° +/-0.029	0.402 +/-0.031	0.444** +/-0.029	0.472** +/-0.040	0.669°° +/-0.032	0.701 +/-0.036	0.743** +/-0.047	0.807** +/-0.046
	Historical control data	-			Historical control data	-

Notes:

Only parameters where statistical significance was demonstrated in one or two sexes are included in the table.

 *= p<0.05; **= p<0.01; Dunnett two-sided test.

°= p<0.05,°°=p<0.01; Analysis of Variance test

Values out of the historical control range are indicated with bold font.

 

Table 2 Haematology (Mean +/- SD)

Sex	Males				Females
Groups/Conc (ppm/day)	Control (0)	Low (1500)	Mid (4200)	High (12500)	Control (0)	Low (1500)	Mid (4200)	High (12500)
Red blood count (M/µL)	9.12 +/-0.402	8.89 +/-0.284	9.01 +/-0.392	8.53 ** +/-0.322	7.821 +/-0.576	8.111 +/-0.407	7.975 +/-0.525	7.931 +/-0.434
	Historical control data	8.07-9.61			Historical control data	-
Haemoglobin concentration (g/dL)	14.85 +/-0.40	14.52 +/-0.51	14.61 +/-0.66	14.21 +/-0.53	14.24° +/-0.58	14.10 +/-0.46	13.91 +/-0.51	13.61 * +/-0.41
	Historical control data	-			Historical control data	13.4-16.3
Mean cell haemoglobin (pg)	16.31 +/-0.57	16.34 +/-0.48	16.21 +/-0.52	16.67 +/-0.47	18.24 +/-1.16	17.39 +/-0.57	17.44 +/-0.73	17.17 * +/-0.53
	Historical control data	-			Historical control data	16.0-19.9
Monocytes (%)	3.19 +/-0.89	3.10 +/-0.51	2.53 +/-0.66	2.55 +/-1.16	3.80° +/-2.06	2.33 *   +/-0.79	2.27 * +/-0.59	2.36 +/-0.48
	Historical control data	-			Historical control data	1.2-6.2
Prothrombin time  (sec)	10.23 +/-0.42	10.07 +/-0.23	10.14 +/-0.29	10.02 +/-0.15	9.69°° +/-0.22	9.50 +/-0.16	9.25 **  +/-0.17	9.35 ** +/-0.14
	Historical control data	-			Historical control data	8.9 – 9.9

Notes:

Only parameters where statistical significance was demonstrated in one or two sexes are included in the table.

*= p<0.05, **= p<0.01; Dunnett two -sided test.

°= p<0.05,°°=p<0.01; Analysis of Variance test

 

Table 3 Clinical chemistry (mean +/-SD)

Sex	Males					Females
Groups/Conc (ppm/day)	Control (0)	Low (1500)		Mid (4200)	High (12500)	Control (0)	Low (1500)	Mid (4200)	High (12500)
ALKP (U/L)	81.2 +/-10.3	89.4 +/-21.0		106.3* +/-20.1	139.0 ** +/-28.6	44.6 +/-14.4	54.4 +/-9.2	62.6* +/-15.7	120.4** +/-36.7
	Historical control data		46 – 128			Historical control data	30 – 66
Bile acids (μmol/L)	9.374 +/-1.269	9.597 +/-2.333		9.367 +/-0.922	11.59^+ +/-1.628	10.910 +/-1.989	10.402 +/-3.061	11.099  +/-2.581	12.653 +/-3.950
	Historical control data		5.53 – 35.56			Historical control data	-
ALT/GPT (U/L)	45.5 +/-7.5	57.2 +/-40.7		45.3 +/-9.3	58.2 +/-22.0	61.3 +/-16.5	47.5* +/-10.2	36.5** +/-2.7	46.8* +/-9.9
	Historical control data		-			Historical control data	21 – 201
K^+ (Potassium) (mmol/L)	6.35 +/-1.09	6.15 +/-0.71		5.66 +/-0.33	6.03 +/-0.58	5.10 +/-0.35	5.35 +/-1.06	5.42 +/-0.32	5.94^++ +/-0.77
	Historical control data		-			Historical control data	4.4 – 6.3

Notes:

Only parameters where statistical significance was demonstrated in one or two sexes are included in the table.

*= p<0.05, **= p<0.01; Dunnett two sided test,

⁺= p<0.05,⁺⁺= p<0.01; Dunn two sided test

Values out of the historical control range are indicated with bold font

 

Table 4 Gross pathology necropsy (Number of affected animals/ number of examined animals) 

 

Sex		Males				Females
Group/conc (ppm/day)		control (0)	Low (1500)	Mid (4200)	High (12500)	control (0)	Low (1500)	Mid (4200)	High (12500)
kidney - dilatation of pelvis	examined	10	10	10	10	10	10	10	10
	normal	10/10	10/10	9/10	9/10	10/10	10/10	10/10	10/10
	dilatation; right, pelvis	0/10	0/10	1/10	1/10	0/10	0/10	0/10	0/10
ovaries	examined	-	-	-	-	10	10	10	10
	normal	-	-	-	-	10/10	10/10	9/10	10/10
	small; bilateral	-	-	-	-	0/10	0/10	1/10	0/10
uterus (cervix+body+horn)	examined	-	-	-	-	10	10	10	10
	normal	-	-	-	-	9/10	9/10	9/10	7/10
	dilatation; body; horn	-	-	-	-	1/10	1/10	1/10	3/10

Table 5 Histopathology (Number of affected animals/number of examined animals)

Sex		Males				Females
Groups/Conc (mg/kg bw/day)		Control (0)	Low (1500)	Mid (4200)	High (12500)	Control (0)	Low (1500)	Mid (4200)	High (12500)
Liver	Examined	10	0	0	10				10
	Hypertrophy, hepatocellular, centrilobular; minimal	0°			8	0			4
	slight	0			1	0			4
	moderate	0			0	0			2
Adrenals	Examined	10	0	0	10	10	0	0	10
	hypertrophy; zona glomerulosa; bilateral, minimal	1			1	0			5
	slight	0			0	0			2
Thyroid gland + parathyroid gland	Examined	10	0	0	10	10	0	0	10
	Hypertrophy, follicular cell; bilateral, minimal	1			6	0			0

 

Notes:

°p<0.001 group factor Chi-Squared & Fisher’s Exact Test: Chi-Squared

ANALYSIS OF THE TREATED DIETS: No test item was detected in the Control samples. The test item was homogenously distributed in each diet. The concentration of the test item in the diets was within the acceptable range at each dose level. Based on these results, the diets were considered suitable for the study purposes.

Table 6: Summary of dose intake values

Group No.	1	2	3	4
Group designation	Control	Low dose	Mid dose	High dose
Nominal dose level (ppm)	0	1500	4200	12500
mean dose intake (mg/kg bw/day)	0
males	0	103	289	895
females	0	111	316	867
combined	0	107	302	881

Applicant's summary and conclusion

Conclusions:

No test item was detected in the control diet samples. The test item was homogenously distributed in each diet. The concentration of the test item in the diets was within the acceptable range at each dose level at all occasions.
Naphthenic acids administered in diet at concentrations of 1500, 4200 and 12500 ppm, was equivalent to dietary doses of approximately 107, 302 and 881 mg/kg bw/day in the Low, Mid and High dose groups, respectively. Reduced palatability of the treated diet was seen at 4200 and 12500 ppm.
In summary, daily administration of Naphthenic acids in diet to Wistar rats during the treatment period under the conditions of this study did not result in test item related mortality or clinical signs. The lower body weight, body weight gain and food consumption values in the Mid and High dose groups are considered to be caused by the low palatability of the treated diet, with no evidence of a clear systemic toxic effect of the test item.
At clinical pathology, increased level of Alkaline Phosphatase activity (ALKP) was recorded in the High dose groups, which may correlate with hepatic effects seen at the organ weight measurement and histopathology. High potassium was measured in some High dose females, corresponding with adrenal histopathology. These findings were not clearly adverse. No other clear test item-related findings were seen in the clinical pathology parameters.
No macroscopic changes were seen at necropsy.Increased liver and kidney weights noted in the High dose male and female animals are considered to be a non-specific adaptive change in the liver and kidney, although the liver weight increases relative to body weight were about 40% and 60% for males and females respectively.At histopathology of the Control and High dose animals, hepatocellular hypertrophy in the liver and follicular cell hypertrophy in the thyroid were seen and are considered as secondary, non-adverse changes. The toxicological significance of hypertrophy in the adrenals, observed mostly in the High dose females, is uncertain.Although none of the individual findings in the High dose group were clearly adverse, taking into account the degree of liver weight increase, and the other findings observed at the High dose, it is considered prudent to not declare the High dose as being free of any adverse findings.
In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for Naphthenic acids is considered to be 302 mg/kg bw/day for the combined sexes (289 mg/kg bw/day for males and 316 mg/kg bw/day for females).