Fatty acids, C6-12, mixed tetraesters with heptanoic acid, pentaerythritol, 3,5,5-trimethylhexanoic acid and valeric acid

Endpoint summary

Description of key information

HATCOL 5236: Subacute 28-day oral toxicity - NOAEL  150 - 1000 mg/kg/day in male and female Wistar rats.

HATCOL 3344: Subacute 28-day oral toxicity - NOAEL 1000 mg/kg/day in male and female Wistar rats.

HATCOL 3331: Subacute 28-day oral toxicity - NOAEL 150 mg/kg/day in male and female Wistar rats.

Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid: Repeated dose toxicity: Oral NOAEL (rat, m/f): = 300 mg/kg bw/day (OECD 408, GLP, analogue approach).

Pentaerythritol ester of pentanoic acids and isononanoic acid: Oral NOAEL (rat, f): 500 mg/kg bw/day (OECD 407, GLP, analogue approach).

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:

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 & 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: United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 71 2-C-00-366, July 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.
Allocation:
Group Dose level number of animals animal numbers
Mg/kg/day males females males females
1 0 (vehicle) 5 5 1-5 21-25
2 50 5 5 6-10 26-30
3 150 5 5 11-15 31-35
4 1000 5 5 16-20 36-40
The dose levels were selected on the basis of a 5-day dose range finding study (NOTOX Project 385392) and in consultation with the sponsor.

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 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:

TEST SUBSTANCE PREPARATION
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:

Once daily for at least 28 days.

Frequency of treatment:

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.

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.

Control animals:

yes, concurrent vehicle

Details on study design:

TREATMENT: 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:

Positive control not used in this study.

Observations and examinations performed and frequency:

OBSERVATIONS
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.
Bodyweights: 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 post mortem 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 mi), with citrate for clotting tests (1.0 ml) and Liheparin treated tubes for clinical biochemistry parameters (0.5 ml).

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.

HISTOTECHNOLOGY: All organ and tissue samples, as defined under Histopathology, 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 specified 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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

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. ,
Bodyweights: Bodyweights 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 - <= 1 000 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; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Key result

Critical effects observed:

not specified

Conclusions:

From the results presented in the study report a definitive No Observed Adverse Effect Level (NOAEL) for HATCOL 5236 of 150 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.

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; bodyweight 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 (female), lower cholesterol values(male).

 

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 150 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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Jan - June 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-guideline with acceptable restrictions (analytical purity not reported, no urinalysis and ophthalmoscopic examination conducted)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 1995

Deviations:

yes

Remarks:

no analytical purity reported, no urinalysis and ophthalmoscopic examination conducted

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

no analytical purity reported, no urinalysis and ophthalmoscopic examination conducted

Qualifier:

according to guideline

Guideline:

other: the Japanese Ministry of Health and Welfare (MHW) Guidelines 1986

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom

Species:

rat

Strain:

other: Sprague-Dawley CD (Crl : CD ® BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, Kent, UK
- Age at study initiation: five to six weeks
- Weight at study initiation: 182-230 g (males), 156-177 (females)
- Housing: animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet: pelleted diet (Rat and mouse SQC Expended Diet No. 1, Special Diets Service Limited, Whitam, UK), ad libitum
- Water: mains water was supplied from polycarbonate bottles attached to cage, ad libitum
- Acclimation period: nine days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

BP

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a solution in arachis oil BP.
VEHICLE
- Concentration in vehicle: 75, 250 and 500 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the test material formulations was determined by gas chromatography (GC) using an external standard technique.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a solution in arachis oil BP.
VEHICLE
- Dose selection rationale: dose levels were based on the results of a foregoing range finding study, in which animals were orally exposed to 150, 500 and 1000 mg/kg bw/day for 28 days (Jones, J., 1998). No adverse effects were observed at throughout. Therefore, 150, 500 and 1000 mg/kg bw/day were selected as the dose levels for the main study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during to working week. Animals were observed immediately before dosing and one hour after dosing at weekends. All records were recorded.
BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Body weights were also recorded at terminal kill.
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was recorded for each cage group at weekly intervals throughout: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
FOOD EFFICIENCY:
- Group mean body weight gain (g/rat/week) / group mean food consumption (g/rat/week) : Yes
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (Day 28) from lateral vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters examined: haemoglobin, erythrocyte count, haematocrit, erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), total leucocyte count (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count (PTL), reticulocyte count (retic), prothrombin time (CT), activated partial thromboplastin time (APTT). Cresyl blue stained slides were prepared but reticulocytes were not assessed.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (Day 28) from lateral vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters examined: urea, calcium (Ca+), glucose Inorganic phosphorus (P), total protein , aspartate aminotransferase (ASAT), albumin alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio, Alkaline phosphatase (AP), Creatinine (Creat), sodium (Na+), total cholesterol (Chol), potassium (K+), total bilirubin (Bili), chloride (Cl-)
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on one occasion prior to the start of treatment and on Days 2, 12, 19 and 27 all animals were observed for signs of functional/behavioural toxicity. On Days 26/27 functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity (grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex, blink reflex)/ grip strength / motor activity / other: behavioural assessment (gait gyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation), lachrymation.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ weights: the following organs removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, kidneys, testes, brain, liver, thymus, epididymides, ovaries, heart and spleen.
HISTOPATHOLOGY: Yes. Sample of the following tissues were removed from all animals and preserved in buffered 10% formalin: adrenals, aorta (thoracic), bone and bone marrow /femur (including stifle joint), bone and bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), urinary bladder, sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, and uterus.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
Haematological, blood chemical, organ weight (absolute and relative to terminal body weight), weekly body weight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous pairwise comparisons were conducted using Dunnetts's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.
Probability values (p) are presented as follows: p < 0.001<, p < 0.01 **, p < 0.05 *, and p ≥ 0.05 (not significant)

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:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths during the study. There were no clinically observable signs of toxicity detected during the study. One male treated with 1000 mg/kg bw/day showed red/brown staining around the eyes on Days 18 and 19 whilst one female showed red/brown staining around the ano-genital region from Day 18 onwards. Such isolated, incidental external changes were regarded as fortuitous and of no toxicological importance.
BODY WEIGHT AND WEIGHT GAIN
No adverse effect on body weight development was detected during the study. A statistically significant reduction in bodyweight gain was detected for 150 mg/kg bw/day females during Week 1 but, in the absence of a convincing dose-related response, the slight intergroup difference was regarded as incidental and of no toxicological significance.
FOOD CONSUMPTION AND COMPOUND INTAKE
A slight reduction in food consumption was detected for females treated with 1000 mg/kg/day throughout the dosing period when compared with controls.
FOOD EFFICIENCY
Food efficiency, however, was similar to that of controls over the same period.
No adverse effect on dietary intake was detected for 1000 mg/kg bw/day males or for animals of either sex treated with 500 or 150 mg/kg bw/day.
WATER CONSUMPTION AND COMPOUND INTAKE
Daily visual inspection of water bottles revealed no intergroup differences.
HAEMATOLOGY
There were no treatment-related changes in the haematological parameters measured. A statistically significant increase in haemoglobin was detected for 1000 mg/kg bw/day males when compared with controls, but, in the absence of any other changes in haematological correlates, this was considered to be fortuitous and of no toxicological significance.
CLINICAL CHEMISTRY
There were no treatment-related changes in the blood chemical parameters measured. A statistically significant increase in plasma inorganic phosphorus was detected for 1000 mg/kg bw/day males when compared with controls but, in isolation, this minimal (p<0.05) intergroup difference was considered not to be toxicologically important.
FUNCTIONAL OBSERVATION
Behavioural assessments
Detailed open-field assessments conducted during the treatment period did not reveal any treatment-related changes in behaviour.
All inter and intra group differences in urination, defecation, and transfer arousal scores were considered to be a result of a normal variation for rats of the strain and age used. Thus these findings were considered to be of no toxicological significance.
Functional performance tests
There were no treatment-related changes in the functional performance parameters measured. Statistical analysis of the data revealed no intergroup differences.
Sensor reactivity assessment
Sensory reactivity assessment did not reveal any treatment-related changes. All inter and intra group differences in sensory scores were considered to be a result of normal variation for rats of the strain and age used and, were therefore, considered being of no toxicological significance.
Statistical analysis of startle reflex data revealed no intergroup differences.
ORGAN WEIGHTS
Animals of either sex treated with 1000 mg/kg bw/day showed a statistically significant increase in liver weight, both absolute and relative to body weight, when compared with controls. The effect extended to 500 mg/kg bw/day animals but statistical significance was only achieved for male relative liver weights. Kidneys weights were also elevated at 1000 and 500 mg/kg bw/day but this was confined to the males and the intergroup difference for 500 mg/kg bw/day group mean absolute kidney weight failed to achieve statistical significance.
No such effects were detected at 150 mg/kg bw/day.
GROSS PATHOLOGY
All of the males treated with 1000 mg/kg bw/day showed speckled kidneys at terminal kill whilst two females from this treatment group showed pallor of the liver.
One female treated with 500 mg/kg bw/day also showed a pale liver at necropsy.
The remaining macroscopic findings including reddened or dark areas on the lungs, hydronephrosis and isolated gastric changes, whilst showing no dose-related response, were consistent with normally expected low incidence findings in laboratory maintained rats and were considered to be of no toxicological importance.
HISTOPATHOLOGY: NON-NEOPLASTIC. Treatment-related kidney changes were observed. Globular accumulations of eosinophilic material were observed in the renal proximal tubular epithelium of males treated at 1000, 500 or 150 mg/kg bw/day. The presence of globular accumulations of eosinophilic material in the tubular epithelium is consistent with the appearance of hydrocarbon nephropathy which results from the excessive accumulation of alpha2-microglobulin in renal proximal tubular epithelial cells. Alpha2-microglobulin is found only in the proximal tubular epithelium of adult male rats, this condition does not, therefore, represent a hazard to human health. All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: The treatment-related effects were confinded to increased liver weight (adaptative response).

Key result

Dose descriptor:

NOAEL

Sex:

male

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Key result

Critical effects observed:

not specified

Conclusions:

Oral administration of the test material to rats for 28 days, at dose levels of up to 1000 mg/kg bw/day resulted in treatment-related changes for males at all dose levels and for females at 1000 mg/kg bw/day. A clear NOAEL for males was not obtained, while the NOAEL for females was considered to be 500 mg/kg bw/day. Nevertheless the treatment-related effects were confined in increased liver weight with no concomitant histopathology and the male rat specific condition, hydrocarbon nephropathy. These changes are not considered to represent serious damage to human health.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Nov 1997 - 16 Mar 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (Dec 2012)", 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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Remarks:

ToxLabs Prüflabor GmbH, Greppin, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation: 32-38 d
- Weight at study initiation: 148.5 g (mean value males), 136.7 g (mean value females)
- Housing: one or two animals in cages (Makrolon Type 3)
- Diet: Altromin 1326, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6-8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 30-60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: distilled water containing 1% Tween 80

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 1%
- Lot/batch no. (if required): S23350 739

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 2 mL samples of each formulation were taken and stored in the frozen state until measurement

Duration of treatment / exposure:

90 d

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10 (control, test and satellite groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for selecting satellite groups: 10 animals each from the high dose and the vehicle group were used to investigate reversibility of possible effects
- Post-exposure recovery period in satellite groups: 28 d

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, autonomic activity, presence of clonic or tonic movements, stereotypies, bizarre behavior
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes, changes in skin, fur, eyes, mucous membranes, gait, posture: response to handling; occurrence of secretions and excretions
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly from the start to the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the administration and at the end of the study
- Dose groups that were examined: All (surviving) animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to killing at the end of the study
- Anesthetic used for blood collection: Yes (Ether)
- Animals fasted: Yes, over night
- How many animals: All animals
- Parameters examined: erythrocyte count, hemoglobin concentration, packed cell volume, platelet count, total leukocyte count, leukocyte differential count, prothrombine time, fibrinogen concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to killing at the end of the study (including the satellite groups)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: alkaline aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, creatinine, fasting glucose, phosphorus, total cholesterol, total protein, albumin, chloride, potassium, sodium

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to administration, at monthly intervals and in the last week of dosing and in the last week of the recovery period.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (auditory, visual and proprioceptive stimuli) / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: cranial, thoracic and abdominal cavities were opened and examined macroscopically
HISTOPATHOLOGY: adrenals, aorta, brain (3 sections), epididymides, eye, femur, heart, kidney, liver, lungs (incl. mainstem bronchi), mesenteric lymph node, muscle incl. sciatic nerve, oesophagus, ovaries, pancreas, pituitary, prostate, seminal vesicle, skin incl. mammary glands, small and large intestine (including peyer´s patches), spinal chord (3 levels), spleen, sternum with bone marrow, stomach, submandibular lymph node, testes, thymus, thyroid (incl. parathyroids), trachea, urinary bladder, uterus

Other examinations:

Organ weights of adrenals, brain, epididymides, testes, heart, kidneys, liver, ovaries, spleen, testes and thymus

Statistics:

Body weights, food consumption: Welch t-test

Haematology, coagulation, clinical biochemistry and absolute and relative organ weights: Dunnett´s test

Differential leukocyte count: Mean, range and standard deviation

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:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

high dose group (female): fibrinogen concentration of plasma and creatinine content increased, high dose group (male/female): alkaline phosphatase increased, middle and high dose groups (male/female): sreum urea nitrogen increased, all non-adverse

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

for high dose males and females, non-adverse

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

intracellular fat and low-grade fatty degenerations of hepatocytes in all male animals, female control, middle dose and high dose groups, non-adverse

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Two animals died shortly after administration due to incorrect gavage shown by lungs filled with blood. None of the animals showed any alterations of their general state of well-being and behaviour at any observation period (few observations were made substance independent and for a short period of time).

BODY WEIGHT AND WEIGHT GAIN
Not affected by the test compound.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Not dose-dependently influenced.

OPHTHALMOSCOPIC EXAMINATION
No alterations.

HAEMATOLOGY
Not influenced.

CLINICAL CHEMISTRY
The fibrinogen concentration of the plasma was increased in the female animals of the high dose group, this was no longer apparent at the end of the treatment-free period.
The activity of alkaline phosphatase of the serum significantly increased in the high dose group, males and femals. This indicates damage to liver cells and/or an increased function rate. This finding was no longer apparent at the end of the treatment-free period.
The serum urea nitrogen was significantly increased in the middle and high dose group of the males and in the high dose group of female animals. The creatinine content was significantly increased in all male and in the high dose group of the female animals. The phosphorus content was significantly increased dose-dependently in all female animals and the sodium content was dose-dependently decreased in the male animals, significantly in the animals of the middle and high dose groups. These effects were no longer apparent at the end of the treatment-free period.

NEUROBEHAVIOUR
No changes in grip strength, motor activity and sensory response.

ORGAN WEIGHTS
Absolute and relative kidney weights were increased in all male animals in the high dose group which was still present after the recovery period. Absolute and relative liver weights were increased in both sexes but this was no longer apparent after the recovery period in females. Other significant differences seem to be incidental.

GROSS PATHOLOGY
No substance-dependent changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Intracellular lipid droplets in hepatocytes of the female animals in the high and mid dose group (5-90% of the observed area) with cell lesions were clearly caused dose-dependently by the test article. There was no special localization of the changes of hepatocytes in the liver lobules. In most cases only low grade intracellular lipopexia occurred in the male animals.
Stomach: Oesophagal part and cardia with multilayered squamous epithelium, leukocyte infiltration in the submucosa and fibrous repair, fibrotic regions in the submucosa of the glandular stomach
Lungs: Atelectactic and emphysemic areas
Thymus: Partial substitution of the parenchyma by fibrinogenesis
Skin: Benign fibrous proliferation
Sciatic nerve: Thickening of the perineurium and thickening of the adventitia of the vessels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Key result

Critical effects observed:

not specified

Conclusions:

The 90-day study with pentaerythritol ester of pentanoic acids and isononanoic acid (CAS 146289-36-3) show the following effects: deposition of intracellular fat and fatty degeneration of the hepatocytes, the increased weights of the liver, both in female only, and the increased activity of alkaline phosphatase correlate with each other and identify the liver as the target organ. However these effects can be seen as adaptive response.
Except for the increased kidney weights in the males, all changes were no longer apparent at the end of the treatment-free period. The increased kidney weights were statistically significant only in the animals of the high dose group. The increase in kidney weights in all male animals could be correlated to the formation of hyaline droplets a phenomenon widely accepted to be specific to the male rat and as such considered to have no relevance to man. The results indicate that the NOAEL was 300 mg/kg bw day.

Executive summary:

A 90-day oral toxicity study with Pentaerythritol ester of pentanoic acids and isononanoic acid (CAS 146289-36-3) was performed according to OECD Guideline 408 and under GLP conditions (Müller, 1998). Groups of 10 male and 10 female Wistar rats were exposed to the substance at 100, 300 and 1000 mg/kg bw/day by gavage daily, 7 days/week for 90 days. Satellite control and high dose groups containing 10 male and female animals each were observed for additional 28 days. Control animals (10 per sex and dose) received the concurrent vehicle, distilled water containing 1% Tween 80. Observations and examinations of the animals included clinical signs, body weight, food consumption, haematology, clinical chemistry, organ weights, neurobehaviour, gross necropsy and histopathology. The daily oral administration of the test substance was tolerated without any adverse effects up to 1000 mg/kg bw/day. No mortality was observed except for two animals that died shortly after administration due to incorrect gavage. Absolute and relative kidney weights were increased in all male animals in the high dose group which was still present after the recovery period. However, histopathology revealed no adverse effects in the kidney. Absolute and relative liver weights were increased in both sexes but this was no longer apparent after the recovery period in females. Other significant differences seem to be incidental. The activity of alkaline phosphatase of the serum significantly increased in the high dose group, males and females. This indicates damage to liver cells and/or an increased function rate. This finding was no longer apparent at the end of the treatment-free period. Except for the increased kidney weights and liver weight in the males, all changes (e.g. clinical chemical changes) were no longer apparent at the end of the treatment-free period.  A 90-day oral NOAEL of 300 mg/kg bw/day was found for Pentaerythritol ester of pentanoic acids and isononanoic acid in male and female rats. However, the increase in kidney weights in all male animals could be correlated to the formation of hyaline droplets a phenomenon widely accepted to be specific to the male rat and as such considered to have no relevance to man.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Remarks:

5 substances available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

see the attached justification in section 13 for the full details.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

Remarks on result:

other: Hatcol 3331

Key result

Dose descriptor:

NOAEL

Effect level:

>= 150 - <= 1 000 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; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Remarks on result:

other: Hatcol 5236

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

Remarks on result:

other: Hatcol 3344

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: The treatment-related effects were confined to increased liver weight (adaptative response).

Remarks on result:

other: Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid

Key result

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Increased liver weight (absolute and relative). Absolute and relative kidney weights were also elevated at 1000 and 500 mg/kg bw. Speckled kidneys. Globular accumulations of eosinophilic material observed in the renal proximal tubular epithelium of males.

Remarks on result:

other: Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

Remarks on result:

other: Pentanoic acid, mixed esters with pentaerythritol, isopentanoic and isononanoic acid (CAS 146289-36-3)

Key result

Critical effects observed:

not specified

Conclusions:

The read across for substance, CASL 156558-98-4; EC: 451-190-; is based upon the analogous substance to which basic form, degree of substitution of fuctional groups is not considered to effect the proposed read across for the endpoint of repeated oral toxicity. The mean NOAEL for the substance is deemed to be 450 mg/kg bw/day based on the information available for readacross.

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June 2003 to 22 July 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU & US EPA testing 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: United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, 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, July 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 21.0 ± 3.0°C (actual range: 18.6-23.7°C), a relative humidity of 30-70% (actual range: 41-87%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Temporary fluctuations from the light/dark cycle (with a maximum of I hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations 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 III, 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 far 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.
Method of formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance (0.97 g/ml) and vehicle.
Storage conditions: At ambient temperature.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Samples of fresh formulations prepared after the in-life phase were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Since a structurally similar Hatcol test substance (HATCOL 5236) was not stable over a 4-hour period in propylene glycol, stability of HATCOL 3331 in propylene glycol was determined over a 1-, 2- and 4-hour period (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 364938).

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily.

Remarks:

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

No. of animals per sex per dose:

ALLOCATION
Group Dose level Number of animals Animal numbers
Mg/kg/day Males Females Males Females
1 0 (vehicle) 5 5 1-5 21-25
2 50 5 5 6-10 26-30
3 150 5 5 11-15 31-35
4 1000 5 5 16-20 36-40

Control animals:

yes, concurrent vehicle

Details on study design:

TREATMENT: 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:

Positive control not used in this study.

Observations and examinations performed and frequency:

OBSERVATIONS
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 (except at the end of week 4), 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, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson 'Technical Services, Debenham, Stowmarket, England).
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 post modem 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).

Sacrifice and pathology:

NECROPSY: All animals surviving to the end of the observation period were deeply anaesthetised using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.
HISTOTECHNOLOGY: All organ and tissue samples, as defined under Histopathology, 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.
Based on the treatment related morphologic changes, kidneys were also examined from all male rats of the intermediate dose groups. All abnormalities were described and included in the report..

Other examinations:

No further examinations specified 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):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

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. Incidental findings that were noted included diarrhoea (one control male), watery discharge from the eye (one male at 50 mg/kg/day), scabs and/or alopecia and the neck (one male and female at 150 mg/kg/day), piloerection (two high dose males on days 8 and 9 only), and a broken tail apex and rales (one female at 150 and 1000 mg/kg/day respectively). At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted among control females and females dosed at 50 mg/kg/day.
Functional Observations: No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with HATCOL 3331, when compared to control animals. The variation in motor activity did not indicate a relation with treatment.
Bodyweights: A slightly lower weight gain was noted for high dose males on day 8 (p<0.01). No further changes were noted regarding bodyweights and bodyweight gain of treated animals over the 4-week study period.
Food Consumption: An increased absolute and relative food intake was noted for high dose males in weeks 1 and 2. Other treated animals showed similar food consumption before or after allowance for bodyweight when compared to control animals.

CLINICAL LABORATORY INVESTIGATIONS
Haematology: Red blood cell count (RBC) and haematocrit levels (HCT) were slightly increased in high dose females (p<0.05). No dose-response relationship was observed for the lower prothrombin time (PT) in males at 50 mg/kg/day, for the increased haemoglobin (HBG) values in females at 50 and 1000 mg/kg/day, for the higher mean corpuscular haemoglobin concentration (MCHC) and partial thromboplastin time (APTT) in females at 50 mg/kg/day, and for the reduced red cell distribution width (RDW) in females at 50 mg/kg/day and above. These changes were considered to be of no toxicological significance.
Clinical Biochemistry: Clinical biochemistry values were similar between control and treated rats.
PATHOLOGY
Macroscopic Examination: Necropsy did not reveal any toxicologically relevant alterations. No microscopic correlate was found for the enlarged liver in one high dose male (no. 17). This was considered to be an incidental finding. Other incidental findings among control and/or treated animals included an accentuated lobular pattern of the liver, pelvic dilation of the kidneys, pale discolouration of the kidneys, red discolouration of the thymus, lungs or mandibular lymph nodes, enlarged mandibular lymph nodes, red foci on the thymus, fluid in the uterus, and a broken tail apex. 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. No findings were noted in control males.
Organ Weights: Liver weights and liver to body weight ratios of high dose females were increased (p<0.05). The increased liver weight and liver to body weight ratio of one high dose male (no. 17) correlated to the macroscopic observation of enlargement (see above). This was considered to be an incidental finding. The statistically significant reduction of thymus to body weight ratio of males at 150 mg/kg/day was considered not to be a sign of toxicity in the absence of a dose response effect. The lower spleen weights and spleen to body weight ratios of females dosed at 50 mg/kg/day and above were considered to be due to slightly higher spleen weights of controls. Similarly, the lower kidney to body weight ratios of females at 150 and 1000 mg/kg/day were considered to have attained statistical significance due to slightly higher kidney weights of control animals. Also, no dose-response relationship was apparent. These changes were considered to be of no toxicological significance.
Microscopic Examination: Treatment-related changes were noted in the kidneys and consisted of:
- Hyaline droplets: 215 males at 50 mg/kg/day, and all males at 150 and 1000 mg/kg/day (minimal to moderate severity);
- Increased incidence and severity of tubular basophilia (slight to moderate): 315 males at 1000 mg/kg/day and 215 males at 150 mg/kg/day;
- Degeneration and/or necrosis of the tubular epithelium of the renal cortex: all males at 1000 mg/kg/day, 415 males at 150 mg/kg/day, and 115 males at 50 mg/kg/day.
All remaining 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; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

Key result

Critical effects observed:

not specified

Conclusions:

From the results presented in the report a No Observed Adverse Effect Level (NOAEL) could not be determined for males, but was established to be 150 mg/kg/day for females. However, since the male kidney findings are a species and sex specific response which is not observed in humans, a NOAEL of 150 mg/kg/day may be considered for both sexes.

Executive summary:

Sub acute 28-day oral toxicity with HATCOL 3331 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 96/541EEC, 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 (71 01), 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; bodyweight 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 4 hours of formulations of test substance in propylene glycol were demonstrated by analyses.

Treatment-related findings observed were as follows:

50 mg/kg/day:- Renal hyaline droplets (2/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (1/5 males).

150 mg/kg/day:- Renal hyaline droplets (all males), increased incidence and severity of tubular basophilia in the kidneys (2/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (4/5 males).

1000 mg/kg/day:- Slightly lower weight gain on day 8 (males).

-lncreased absolute and relative food intake in weeks 1/2 (males).

-Slightly increased red blood cell count and haematocrit levels (females).

-lncreased liver weights and liver to bodyweight ratios (females).

-Renal hyaline droplets: (all males), increased incidence and severity of tubular basophilia in the kidneys (3/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (all males).

 

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) could not be determined for males, but was established to be 150 mg/kg/day for females. However, since the male kidney findings are a species and sex specific response which is not observed in humans, a NOAEL of 150 mg/kg/day may be considered for both sexes.

Reference 6

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2003 to 15 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU & 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 (7101), 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.

ALLOCATION
Group Dose level Number of animals Animal numbers
Mg/kg/day Males Females Males Females
1 0 (vehicle) 5 5 1-5 21-25
2 50 5 5 6-10 26-30
3 150 5 5 11-15 31-35
4 1000 5 5 16-20 36-40
The dose levels were selected on the basis of a 5-day dose range finding study (NOTOX Project 385324).

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 21.0 ± 3.0°C (actual range: 18.6 - 24.2°C), a relative humidity of 30-70% (actual range: 41 - 88%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations 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 III, height 15 cm.) with sterilised sawdust 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.
Method of formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance and vehicle.
Storage conditions: At ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of week 4 formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Since a structurally similar Hatcol test substance (HATCOL 5236) was not stable over a 4-hour period in propylene glycol, stability of HATCOL 3344 in propylene glycol was determined over a I-2,- and 4-hour period (highest and lowest concentration). Based on the analytical results, analyses were repeated for group 2 (accuracy, homogeneity and stability over 4 hours) and group 3 (accuracy) after the in-life phase. For re-analysis, a sample volume of 1 ml instead of 0.5 ml was used, so that analytical results would more accurately reflect the actual dose concentrations as small droplets were noted in the formulations. Also, the higher sample volume would better approximate the actual dose volumes administered to the animals.
The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX project 364951).

Duration of treatment / exposure:

28 days

Frequency of treatment:

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.

Remarks:

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

No. of animals per sex per dose:

5 males and 5 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

TREATMENT: 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 bodyweight.

Positive control:

Postive control not used in this study.

Observations and examinations performed and frequency:

OBSERVATIONS
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 (inadvertently, no arena observations were performed on days 22 and 28). 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, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Bodyweights: 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.

Sacrifice and pathology:

CLINICAL LABORATORY INVESTIGATIONS: Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled post modem 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 Li-heparin treated tubes for clinical biochemistry parameters (0.5 ml).

NECROPSY: All animals surviving to the end of the observation period were deeply anaesthetised using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.

HISTOTECHNOLOGY: All organ and tissue samples, as defined under Histopathology, 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.

Other examinations:

No further examinations specified 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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

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. Incidental findings that were noted included scabs, alopecia, chromodacryorrhoea and broken upper incisors. These findings are more frequently 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. No clinical signs were noted in control males, females dosed at 50 mg/kg/day, animals dosed at 150 mg/kg/day and high dose males.
Functional Observations: No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with HATCOL 3344, when compared to control animals. Variations noted in motor activity data between treated and control animals occurred in the absence of a dose-related response, similar changes of low or high sensor values, and/or supportive clinical signs. Therefore, they were considered to be of no toxicological relevance.
Bodyweights: No toxicologically relevant changes were noted in body weights and body weight gain of treated animals. Minimal weight loss was noted for one control female (no. 24) and one female dosed at 50 mg/kg/day (no. 26) at the end of week 4. A slightly lower mean weight gain was noted for all treated males in weeks 3/4, while weight gain was also slightly lower for females at 150 mg/kg/day over week 3. These changes occurred in the absence of a dose-response relationship and were therefore considered to be of no toxicological significance.
Food Consumption: The variation noted in food consumption before or after allowance for body weight between treated and control animals was within the normal range for this type of study.

CLINICAL LABORATORY INVESTIGATIONS
Haematology: No toxicologically relevant changes occurred in haematological parameters of treated rats. White blood cell counts (WBC) of high dose females were reduced. This was considered to be mainly due to a low value for one female (no. 40). The remaining two values for this group were within the normal range for this type of study. Therefore, the deviation was considered to be incidental in nature. Values of other haematological parameters between treated and control animals were similar.
Clinical Biochemistry: Alanine aminotransferase activity values (ALAT) were increased in high dose females (p<0.05). A high aspartate aminotransferase activity value (ASAT) was noted in one male dosed at 50 mg/kg/day (no. 7). Values of other animals of this dose group were similar to control values. The statistically significant increase of total protein and albumin levels of females at 150 mg/kg/day occurred in the absence of a dose-response relationship. No toxicological relevance was ascribed to these changes.

PATHOLOGY
Macroscopic Examination: Necropsy did not reveal any toxicologically relevant alterations. Incidental findings among control and/or treated animals included pelvic dilation of the kidneys, red discolouration of the thymus, mandibular lymph nodes or ovaries, right lateral lobe of the liver grown together with the diaphragm, yellowish-soft nodules on the epididymides, diaphragmatic hernia of the right lateral lobe of the liver, a constricted spleen, red foci on the mandibular lymph nodes, fluid in the uterus, exophthalmus and watery-clear cysts on the ovaries. 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:body weight ratios of treated animals were 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:

1 000 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; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

Key result

Critical effects observed:

not specified

Conclusions:

Based on the results presented in the report, a NOAEL of 1000 mglkglday for HATCOL 3344 was established.

Executive summary:

Sub-acute 28-day oral toxicity with HATCOL 3344 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 96/54/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; bodyweight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS

Accuracy and homogeneity of formulations of test substance in propylene glycol were acceptable. Group 2 formulations (50 mg/kg/day) were not stable over a 4-hour period (approximately 50% recovery), while stability of group 4 formulations (1000 mg/kg/day) was confirmed over the same time-period. Based on overall results and the established NOAEL, it was considered that sufficient analytical support was obtained for the purpose of this study.

Treatment-related findings observed were as follows:

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

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

-1000 mg/kg/day: No treatment-related findings noted.

CONCLUSION: Based on the results presented in this report, a NOAEL of 1000 mg/kg/day for HATCOL 3344 was established.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1-2

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

HATCOL 5236:

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 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; bodyweight 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 (female), lower cholesterol values (male).

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 150 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.

HATCOL 3344:

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 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; bodyweight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS: Accuracy and homogeneity of formulations of test substance in propylene glycol were acceptable. Group 2 formulations (50 mg/kg/day) were not stable over a 4-hour period (approximately 50% recovery), while stability of group 4 formulations (1000 mg/kg/day) was confirmed over the same time-period. Based on overall results and the established NOAEL, it was considered that sufficient analytical support was obtained for the purpose of this study.

Treatment-related findings observed were as follows:

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

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

-1000 mg/kg/day: No treatment-related findings noted.

CONCLUSION: Based on the results presented in this report, a NOAEL of 1000 mg/kg/day for HATCOL 3344 was established.

HATCOL 3331:

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 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; bodyweight 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 4 hours of formulations of test substance in propylene glycol were demonstrated by analyses. Treatment-related findings observed were as follows:

50 mg/kg/day:- Renal hyaline droplets (2/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (1/5 males).

150 mg/kg/day:- Renal hyaline droplets (all males), increased incidence and severity of tubular basophilia in the kidneys (2/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (4/5 males).

1000 mg/kg/day:- Slightly lower weight gain on day 8 (males). -lncreased absolute and relative food intake in weeks 1/2 (males). -Slightly increased red blood cell count and haematocrit levels (females). -lncreased liver weights and liver to bodyweight ratios (females). -Renal hyaline droplets: (all males), increased incidence and severity of tubular basophilia in the kidneys (3/5 males), degeneration and/or necrosis of the tubular epithelium of the renal cortex (all males).

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) could not be determined for males, but was established to be 150 mg/kg/day for females. However, since the male kidney findings are a species and sex specific response which is not observed in humans, a NOAEL of 150 mg/kg/day may be considered for both sexes.

Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid

One 28- day study conducted PE/TMP tetra/tri C5, i-C5, C7, C8, i-C9, C10 ester showed no signs of overt toxicity except for treatment-related kidney changes. Globular accumulations of eosinophilic material were observed in the renal proximal tubular epithelium of males treated at 1000, 500 or 150 mg/kg bw/day. The presence of globular accumulations of eosinophilic material in the tubular epithelium is consistent with the appearance of hydrocarbon nephropathy which results from the excessive accumulation of alpha2-microglobulin in renal proximal tubular epithelial cells. Alpha2-microglobulin is found only in the proximal tubular epithelium of adult male rats, this condition does not, therefore, represent a hazard to human health. All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance. For females, treatment related effects were observed at 1000 mg/kg bw/day. A clear NOAEL for males was not obtained, while the NOAEL for females was considered to be 500 mg/kg bw/day. 

Pentaerythritol ester of pentanoic acids and isononanoic acid

The 90-day study with pentaerythritol ester of pentanoic acids and isononanoic acid (CAS 146289-36-3) show the following effects: deposition of intracellular fat and fatty degeneration of the hepatocytes, the increased weights of the liver, both in female only, and the increased activity of alkaline phosphatase correlate with each other and identify the liver as the target organ. However these effects can be seen as adaptive response.

Except for the increased kidney weights in the males, all changes were no longer apparent at the end of the treatment-free period. The increased kidney weights were statistically significant only in the animals of the high dose group. The increase in kidney weights in all male animals could be correlated to the formation of hyaline droplets a phenomenon widely accepted to be specific to the male rat and as such considered to have no relevance to man.

The results indicate that the NOAEL was 300 mg/kg bw day.

 

Justification for classification or non-classification

The substances available for read across have not been classified for specific target organ toxicity in accordance with regulation (EC) 1272/2008, therefore the substance subject to registration is not expected to meet the criteria for classification.