Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

Endpoint summary

Description of key information

RA-S, CAS 68424-31-7, Key, Croda, Brammer, 1993, rep. dose, 28 d, oral, RL2 - NOAEL 1450 mg/kg bw/d

Subacute 28 day oral toxicity- NOAEL 150 mg/kg/day male/female rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov. 1992 - Dec. 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance CAS 68424-31-7. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alpk:APfSD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zeneca Pharmaceuticals, Alderly Park, Macclesfield, Cheshire, UK
- Age at study initiation: 28 d
- Weight at study initiation: Males: 148.45 g; Females: 122.6 g
- Housing: sexes separately, five per cage, Cages had measurements of 26.5x50.0x20.0 cm and were constructed of stainless steel mesh with one solid side.
- Diet: ad libitum (Based on CT1 diet; Special Diets Services Limited, Witham, Essex, UK
- Water: ad libitum
- Acclimation period: approx. 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 45-65 (71 at one occasion)
- Air changes (per hr): 25-30
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: November 1992 To: December 1992

Route of administration:

oral: feed

Vehicle:

other: ethyl acetate

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: All diet were based on CT1 diet (Special Diets Services Limited, Witham, Essex, UK). They were prepared by grinding the appropriate amount of test substance with 1 kg of milled CT1 diet. This premix was then added to 14 kg of diet and mixed thoroughly with a Pharma Blender Model PMA 100S (T K Filder).

DIET PREPARATION
- Rate of preparation of diet (frequency): 15 kg batches
- Mixing appropriate amounts with (Type of food): CT1 diet (Special Diets Services Limited, Witham, Essex, UK)
- Storage temperature of food: - 20°C, stored at RT for usage up to 14 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical stability was determined for diets over a period of 5 weeks following storage at RT or at -20°C.
Samples were extracted by chemical shaking with ethyl acetate. The supernatant was diluted with ethyl acetate to give solutions containing appropriate concentrations of the test substance. Extracts were analysed by gas chromatography using flame ionisation detection. The extract concentration was calculated by reference to data from a standard containing a known concentration.

Duration of treatment / exposure:

daily

Frequency of treatment:

28 d

Remarks:

Doses / Concentrations:
0 ppm, 1000 ppm, 5000 ppm, 12500 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
112 mg/kg/d, 562 mg/kg/d, 1450 mg/kg/d
Basis:
other: actual ingested for males

Remarks:

Doses / Concentrations:
119 mg/kg/d, 586 mg/kg/d, 1613 mg/kg/d
Basis:
other: actual ingested for females

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on results of preliminary feeding studies
- Rationale for animal assignment (if not random): The sexes were randomised separately.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: changes in clinical condition and behaviour and significant changes were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on days 8, 15, 22, 29
- observations included, but were not limited to the assessment of autonomic function (e.g. lachrymation, salivation, piloerection, exophthalmus, urination, defecation, pupillary function, ptosis), description, incidence and severity of any convulsions, tremors, abnormal motor function, alteration in respiration, reactivity to stimuli, changes in the level of arousal, sensorimotor responses

BODY WEIGHT: Yes, measurement in replicate order immediately before feeding and at the same day once a week until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight: Yes, on a weekly basis
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes: At termination, all rats were bled by cardiac puncture and samples were collected. Parameters determined: Hemoglobin, red cell count, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, platelet count, white blood cell count, neutrophil count, lymphocyte count, monocyte count, eosinophil count, prothrombin time and kaolin-cephalin time

CLINICAL CHEMISTRY: Yes, At termination, all rats were bled by cardiac puncture and samples were collected. Parameters determined: Albumin, total protein, cholesterol, triglycerides, urea, creatinine, glucose, total bilirubin and alkaline phosphatase, plasma gamma-glutamyl transferase, plasma alanine aminotransferase, plasma aspartate aminotransferase, plasma creatine kinase, plasma sodium, plasma potassium, plasma chloride, plasma calcium and plasma phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on days 8, 15, 22, 29
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (Adrenals, Aorta, Bladder, Bone and Bone marrow (femur), Brain, Caecum, Colon, Cervical lymph node, Cervix, Colon, Duodenum, Epididymis, Eye and harderian gland, Heart, Ileum, Jejunum, Kidney, Liver, Lungs, Mammary gland, Mesenteric lymph node, Nasal passages, Oesophagus, Oral cavity, Ovaries, Pancreas, Parathyroid glad, Pituary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skin, Spinal chord, Spleen, Sternum, Stomach, Testes, Thymus, Thyroid gland, Trachea, Uterus, Voluntary muscle)

Statistics:

Bodyweights were considered by analysis of covariance on initial body weight, separately for males and females.
Time to tail flick and fore and hindlimb grip strength at weeks 2, 3, 4 and 5 were considered by analysis of variance, separately for both sexes.
Haematological and clinical blood parameters were considered by analysis of variance.
Organ weights were considered by analysis of variance and covariance on final body weight separately for both sexes.

Details on results:

DIET ANALYSIS:
All diets prepared were found to be within 4 % of the target concentration. The homogeneity of the test material in the diet, determined at 1000 and 12500 ppm inclusion levels was within 2 % of the overall mean concentration for both levels. Chemical stability of the test material , assessed at the 1000 and 12500 ppm inclusion levels stored at room temperature or at -20 °C was satisfactory over the period of use.
Dose rates (based on nominal dietary levels) were highest at the start of the study and declined rapidly during the period of rapid growth to week 4.

MORTALITY
There were no mortalities.

FUNCTIONAL OBSERVATION BATTERY
There were no mortalities.
A slightly reduced splay reflex was observed in one female on the 1000 ppm group (on days 29 and 30), one male in the 5000 ppm group (on day 29) and one male in the 12500 ppm group (on day 29). As isolated observations, these were considered to be incidental to treatment.
There were no differences in time to tail flick in either sex which could be attributed to treatment. The statistically significant increase in time to response observed on day 22 for males (5000 ppm) and day 8 for females (1000 ppm) were considered to be incidental to treatment in the absence of similar changes at higher dose levels.
There was no evidence of any treatment related effects on forelimb or hindlimb grip strength.
Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant effects on body weight and all final bodyweights were within 3% of the respective controls, after adjusting for initial weight differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption in all treated groups remained similar to, or exceeded that, of the respective control group throughout the study.

HAEMATOLOGY
There were statistical significant reductions in haemoglobin and haematocrit at 12500 ppm in male rats. Statistically significant reductions in haemoglobin and haematocrit were seen in females at 1000 and 5000 ppm and in white blood cell count at 1000 ppm. In the absence of a coherent dose-response relationship, these differences were considered incidental to treatment.
Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

CLINICAL CHEMISTRY
There were minor reductions in plasma cholesterol, triglyceride and total protein levels and plasma alanine transferase activities in males at 12500 ppm compared to controls. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.
ORGAN WEIGHTS
Kidney weights adjusted for body weight were statistically significant increased in males at 5000 and 12500 ppm. All the females in the treatment groups had slightly raised kidney weights compared to control, but none achieved statistical significance, and there was no evidence of a coherent dose response relationship.
Liver weights adjusted for body weight were statistically significant increased in both sexes at 12500 ppm and in males at 5000 ppm.
Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

PATHOLOGY:
Macroscopic findings:
No treatment-related macroscopic findings were apparent at the end of the study.
Microscopic findings:
Treatment related findings were present in the kidney of male rats from all dose groups. In the 5000 and 12500 ppm dose group these comprised increased tubular hyaline droplet formation and tubular basophilia in all animals, and granular cast formation in four of the 5000 ppm animals and all of the 12500 ppm animals; the latter occurring at the cortico-medullary injection. In the 1000 ppm group, increased renal hyaline droplet formation and/or tubular basophilia were seen, but not granular cast formation.
In the liver, there was minimal hepatocyte hypertrophy in four out of five male rats in the 12500 ppm group.

The increased kidney weights and microscopic findings of renal tubular basophilia, granular cast formation and increased hyaline droplet formation present in male rats at 5000 and 10000 ppm are clearly treatment related. These findings are consistent with the well characterized light hydrocarbon nephropathy described for male rats, following to a variety of chemicals including light hydrocarbons such as unleaded gasoline and trimethyl pentane. The characteristics include an increased accumulation of hyaline droplets in male rat kidneys, the main constituent of which is alpha 2µ-globulin (Alden et al. Adv. Modern Environ Toxicol 7: 107-120 (1984); Stonard et al. Renal Heterogeneity and Target Cell Toxicity. Bach PH and Lock EA Eds, John Wiley and Sons (1985)). It is widely accepted that this phenomenon is specific to male rat and as such appears to have no relevance for man (Swenberg et al. Toxicol and App. Pharmacol. 97: 35-46 (1989)).

Key result

Dose descriptor:

NOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects observed in female rats

Key result

Dose descriptor:

NOAEL

Effect level:

1 613 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects observed in female rats

Key result

Dose descriptor:

NOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Treatment related histopathological changes and changes in kidney and liver weight in male rats at 5000 ppm and above are considered species-specific effects which are not relevant for humans and therefore not considered for NOAEL determination.

Key result

Dose descriptor:

NOAEL

Effect level:

1 450 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Treatment related histopathological changes and changes in kidney and liver weight in male rats at 5000 ppm and above are considered species-specific effects which are not relevant for humans and therefore not considered for NOAEL determination.

Key result

Critical effects observed:

not specified

Conclusions:

Repeated dietary administration of the test material to rats, up to and including a dose level of 1450 mg/kg bw/day for male rats and 1613 mg/kg bw/d for female rats, did not produce any evidence of overt toxicity. There were no clinical signs indicative of neurological dysfunction or neuropathological changes in the brain related to treatment with the test material at any dose level.
In male rats, an increased incidence of renal hyaline droplet formation and tubular basophilia was present at all dose levels, with granular formation and increased kidney weights also present at 5000 and 12500 ppm. This phenomenon is believed to be specific to male rats as such and is not considered to be of relevance to man.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

The substance is a member of a group of pentaerythritol and a mixture of alkyl carboxylic acids which share similar characteristics across the group. Please see attached justification for read across.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption; water consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Key result

Dose descriptor:

NOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

female

Remarks on result:

other: CAS 68424-31-7

Key result

Dose descriptor:

NOAEL

Effect level:

1 613 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

other: CAS 68424-31-7

Key result

Dose descriptor:

NOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Treatment related histopathological changes and changes in kidney and liver weight in male rats at 5000 ppm and above are considered species-specific effects which are not relevant for humans and therefore not considered for NOAEL determination.

Remarks on result:

other: CAS 68424-31-7

Key result

Dose descriptor:

NOEL

Effect level:

1 450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Treatment related histopathological changes and changes in kidney and liver weight in male rats at 5000 ppm and above are considered species-specific effects which are not relevant for humans and therefore not considered for NOAEL determination.

Remarks on result:

other: CAS 68424-31-7

Key result

Critical effects observed:

not specified

Conclusions:

The substance, CAS 68441-67-8, is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of repeated dose toxicity via the oral route. The substance is considered to be not systemically toxic for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Executive summary:

The substance, CAS 68441-67-8, is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of repeated dose toxicity via the oral route. The substance is considered to be not systemically toxic for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 July 2003 to 04 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU and US EPA test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (71 Ol), EPA 712-C-00-366, 2000.

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System: Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EC). Source: Charles River Deutschland, Sulzfeld, Germany.
Age at Start of Treatment: Approximately 6 weeks.
Number of animals: 20 males, 20 females (females were nulliparous and non-pregnant).
Randomisation: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification: Earmark and tattoo.

Conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21 ± 3°C (actual range: 18.6 - 23.7°C) a relative humidity of 30 - 70% (actual range: 41 - 81 %) and 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal level of 70% for relative humidity. Based on laboratory historical data these conditions were considered not to have affected the study integrity.
Accommodation: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type Ill, height 15 cm.) with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding. Results of bedding analyses for contaminants are examined and archived.
Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
Water: Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
Analysis of bedding, diet and water did not reveal any findings that were considered to have affected study integrity.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

Vehicle: Propylene glycol, specific gravity 1.036
Rationale for vehicle: Based on trial formulations performed at NOTOX.
Stability of test substance in vehicle: At least 1 hour (determined at NOTOX).
Method of formulation: Initially, formulations (w/w) were prepared daily within 4 hours prior to dosing. However, results from stability analyses showed that formulations were stable for at least 1 hour at room temperature. Therefore, from 21 July (beginning of week 3) onwards formulations were prepared within approximately 1.25 hours prior to dosing. Formulations were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.
Storage conditions: At ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of week 2 formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 4 hours was also determined (highest and lowest concentration). After the in-life phase additional analyses were performed to check accuracy of preparation (all dose groups) and stability over 1 and 2 hours (highest and lowest concentration). The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX Project 364949).

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily.

Remarks:

Doses / Concentrations:
0, 50, 150, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females per dose group (20 males and 20 females in total).

Control animals:

yes, concurrent vehicle

Details on study design:

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
Method: Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing.
Frequency: Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.
Dose volume: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Positive control:

Postive control not required in this study.

Observations and examinations performed and frequency:

Mortality / Viability: At least twice daily.
Clinical signs: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe
Functional Observations: During week 4 of treatment, motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England) was performed on all animals.
Body weights: On days 1,8, 15,22 and 28.
Food consumption: Weekly.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

CLINICAL LABORATORY INVESTIGATIONS: Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled postmortem examination, between 7.30 and 9.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (1.0 ml) and Liheparin treated tubes for clinical biochemistry parameters (0.5 ml).Parameters determined reported in table form – attached under Any other information.

Sacrifice and pathology:

NECROPSY: All animals were deeply anaesthetised the end of the observation period using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution:
Identification marks: not processed, Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, (Cervix), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, (Seminal vesicles), (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina), All gross lesions.

ORGAN WEIGHTS: The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOTECHNOLOGY: All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY: The following slides were examined by a pathologist:
- all tissues and organs collected at the scheduled sacrifice from all animals of the control and the highest dose group;
- all gross lesions of all animals.
All abnormalities were described and included in the report. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement.

Other examinations:

No further examinations detailed in the study report.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one-t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. - The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on motor activity data. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Red blood cell count (RBC) was increased in females at 1000 mg/kg/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cholesterol values were reduced in males at 1000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlargement of the liver was noted in two males at 1000 mg/kg/day.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Analysis of Dose Preparations: Analyses of HATCOL 5236 in propylene glycol were based on two peaks observed in the GCMS chromatogram. Test substance formulations formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 88% and 119% of target. Although a few values were slightly outside the nominal range of 100 ± 10%, mean values were within this range.
Results from additional analyses of formulations revealed accuracy values between 103% and 120%. No explanation was found for these slightly higher values. Based on the overall accuracy results, it was concluded that an acceptable level of accuracy for this study was achieved.
Group 4 formulations were stable for at least 2 hours at room temperature, and analyses of these formulations after 4 hours storage at room temperature showed results slightly below the limit of 90% (i.e. 89%). Stability analyses of group 2 formulations showed that the formulations were stable for at least 1 hour at room temperature. However, stability measurements of these formulations showed a recovery of 63% and 71 % after a 2-hour recovery period and a recovery of 58% and 65% after a 4-hour period. Based on these results it was decided to reduce the time between formulating and dosing to a maximum of 1.25 hours, starting at the beginning of week 3. On a few occasions during the study time between preparation of the formulations and dosing (slightly) exceeded 1.25 hours.
The No Observed Adverse Effect Level (NOAEL) in this study was established at 1000 mg/kg/day and stability of formulations of 1000 mg/kg/day over 4 hours was only slightly below the acceptable limit. Therefore, it was concluded that sufficient evidence that animals were dosed at least very close to the target dose of 1000 mg/kg was available and that sufficient analytical support was obtained for the purpose of this study.

OBSERVATIONS
Mortality: No mortality occurred during the study period.

Clinical Signs: There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment.
Greasy coat and red discolouration of the fur was noted in two females treated at 1000 mg/kg/day on two days during the third treatment week. Since these findings were only incidental, these effects were considered not to be treatment related. Other incidental findings that were noted included alopecia and watery discharge from the eye. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

Functional Observations: The variation in motor activity did not indicate a relation with treatment.

Body Weights: Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

Food Consumption: Food consumption before or after allowance for body weight was similar between treated and control animals.

CLINICAL LABORATORY INVESTIGATIONS
Haematology: Red blood cell count (RBC) was increased in females at 1000 mg/kg/day.
The decrease of partial thromboplastin time (APTT) in males at 150 mg/kg/day and the decrease of prothrombine time (PT) in females at 150 and 1000 mg/kg/day was considered not to be toxicologically relevant considering the direction of the change (i.e. a decrease) and/or the lack of an effect at the highest dose level.
In females, the lower white blood cell count and higher neutrophil count at 50 and 150 mg/kg/day occurred in the absence of a dose-response relationship and therefore considered not to be related to treatment with HATCOL 5236.

Clinical Biochemistry: Cholesterol values were reduced in males at 1000 mg/kg/day.
Changes in creatinine, total protein values, calcium concentration and albumin concentration in males at 50 and 150 mg/kg/day, respectively were considered to be of no toxicological significance in the absence of a treatment-related distribution.

PATHOLOGY
Macroscopic Examination: Enlargement of the liver was noted in two males at 1000 mg/kg/day.
Incidental findings among control and treated animals included an accentuated lobular pattern of the liver, pelvic dilation of the kidneys, reduced size of testes and epididymides, reddish or red foci on thymus, red discolouration of the thymus and of the mandibular lymph nodes, caecum and colon distended with gas, adrenals grown together with kidneys and fluid in the uterus. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

Organ Weights: Organ weights and organ to body weight ratios of treated animals were considered to be similar to those of control animals.

Microscopic Examination: There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption; water consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Key result

Critical effects observed:

not specified

Conclusions:

No Observed Adverse Effect Level (NOAEL) for HATCOL 5236 of 150 mg/k/day was established. Based on the absence of functional or morphological disturbances supporting higher red blood cell count and lower cholesterol values in the high dose females and males respectively, a NOAEL of 1000 mg/kg/day may be considered.

Executive summary:

Subacute 28-day oral toxicity with HATCOL 5236 by daily gavage in the rat.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day.

 

The study was based on the following guidelines:EC Directive 96154/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996. OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 1995. OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

 

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats.

One control group and three treated groups were tested, each consisting of 5 males and 5 females.

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

 

RESULTS

Accuracy, homogeneity and stability over at least 1 hour (50 mg/kg formulations) or at least 2 hours (1000 mg/kg formulations) of formulations of test substance in propylene glycol were demonstrated by analyses.

50 mg/kg/day: No treatment-related findings noted.

150 mg/kg/day: No treatment-related findings noted.

1000 mg/kg/day: Higher red blood cell count (F), lower cholesterol values(M).

 

CONCLUSION

The higher red blood cell count and the lower cholesterol values found in the high dose females and males respectively, were not supported by other changes in blood parameters or histopathological lesions. Therefore, the toxicological relevance of these effects is doubtful. An enlarged of the liver was noted in two males at 1000 mg/kg/day. Since this was not seen in the other high dose animals and since no morphological correlates were found, this finding was considered not to be toxicologically relevant.

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for HATCOL 5236 of 1 50 mg/kg/day was established. Based on the absence of functional or morphological disturbances supporting higher red blood cell count and lower cholesterol values in the high dose females and males respectively, a NOAEL of 1000 mg/kg/day may be considered.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Fatty acids, C5-10, esters with pentaerythritol (CAS No.: 68424-31-7), were administered in concentrations of 0 ppm, 1000 ppm, 5000 ppm, 12500 ppm (resembling 112 mg/kg bw/d, 562 mg/kg bw/ d and 1450 mg/kg bw/d for male and 119 mg/kg bw/d, 586 mg/kg bw/d and 1613 mg/kg bw/d for female rats, respectively) to 5 animals per sex and dose for 28 consecutive days. There were no toxicologically significant effects on body weight, food consumption and clinical condition up to and including the highest dose level. Changes in some clinical chemistry and red cell-related parameters were observed in male rats at 12500 ppm, but these were minor and considered not to be of toxicological significance. There were no clinical signs indicative of neurological dysfunction in any of the treatment groups, nor was there any evidence of neuropathological changes in the brains of the 12500 ppm group. A minimal hepatocyte hypertrophy, present in males in the 12500 ppm group, is considered to be evidence of an adaptive response. Microscopic examination of the kidneys from male animals from all dose groups revealed an

increase in hyaline droplet formation (the main constituent of which is alpha-2μ-globulin) and tubular basophilia; this phenomenon is widely accepted to be specific to the male rat and as such is considered to have no relevance to man. A NOAEL of 1450 and 1613 mg/kg/d could be identified for male and female rats, respectively (RA-S, CAS 68424-31-7, Key, Croda, Brammer, 1993, rep. dose, 28 d, oral, RL2).

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day.

 

The study was based on the following guidelines:EC Directive 96154/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996. OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 1995. OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

 

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats.

One control group and three treated groups were tested, each consisting of 5 males and 5 females.

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

 

RESULTS

Accuracy, homogeneity and stability over at least 1 hour (50 mg/kg formulations) or at least 2 hours (1000 mg/kg formulations) of formulations of test substance in propylene glycol were demonstrated by analyses.

50 mg/kg/day: No treatment-related findings noted.

150 mg/kg/day: No treatment-related findings noted.

1000 mg/kg/day: Higher red blood cell count (F), lower cholesterol values(M).

 

CONCLUSION

The higher red blood cell count and the lower cholesterol values found in the high dose females and males respectively, were not supported by other changes in blood parameters or histopathological lesions. Therefore, the toxicological relevance of these effects is doubtful. An enlarged of the liver was noted in two males at 1000 mg/kg/day. Since this was not seen in the other high dose animals and since no morphological correlates were found, this finding was considered not to be toxicologically relevant.

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for HATCOL 5236 of 1 50 mg/kg/day was established. Based on the absence of functional or morphological disturbances supporting higher red blood cell count and lower cholesterol values in the high dose females and males respectively, a NOAEL of 1000 mg/kg/day may be considered.

Justification for classification or non-classification

Based on the available information the substance is not classified as systemically toxic, in accordance with Regulation (EC) 1272/2008.