Tall oil, compd. with ethanolamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 August 2012 to 12 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, compd. with ethanolamine

EC Number:

268-069-1

EC Name:

Tall oil, compd. with ethanolamine

Cas Number:

68002-54-0

Molecular formula:

Not applicable. It is not considered possible to assign a specific molecular formula to a UVCB substance.

IUPAC Name:

Tall oil, compd. with ethanolamine

Test material form:

liquid: viscous

Details on test material:

- Appearance: Amber/dark orange viscous liquid/fluid gel
- Storage conditions: Room temperature, protected from light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han)
- Age at study initiation: On the first day of dosing, animals were 71 to 78 days of age.
- Weight at study initiation: On the first day of dosing, the males weighed 283 g to 333 g and the females weighed 184 g to 219 g.
- Housing: The animals were housed in groups of 5 by sex until pairing and for males post-pairing. For pairing, one male and one female were housed together and mated females were housed individually during gestation and with their litter during the lactation period. Males were housed in grid-floor cages suspended over paper-lined trays. During the pre-pairing and mating periods females were housed in grid-floor cages suspended over paper-lined trays and following mating females and their litter were housed in solid-floor cages with appropriate bedding provided.
- Diet (e.g. ad libitum): A pelleted rodent diet was supplied ad libitum.
- Water (e.g. ad libitum): Mains tap water (in bottles) was freely available.
- Acclimation period: The animals were acclimatised within the study room for 14 days before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C (target range 21 ± 2 °C).
- Humidity (%): 46 to 79 % (target range 55 ± 15 %).
- Air changes (per hr): Not reported - the room was air-conditioned.
- Photoperiod (hrs dark / hrs light): The study room was illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dark.

IN-LIFE DATES: From: 2 August 2012 To: 12 November 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Elga UHP water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The required amount of test material was weighed and approximately 80 % of the vehicle was added. The mixture was stirred and heated to approximately 50 ± 5 °C, as required, until a visibly homogenous liquid was formed. The formulation was allowed to cool to ambient temperature, followed by further addition with the vehicle to the exact volume. Due to the lack of any stability information, the formulations were prepared daily and used within 3 hours of preparation.

VEHICLE
- Dose volume: 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

FORMULATION SAMPLING
Samples were taken from each formulation. The formulations (including controls) prepared for use on the first day of dosing and subsequent formulations prepared for use during Week 6 were analysed to assess achieved concentrations. All remaining samples were stored frozen (approximately -18 °C).

ANALYSES OF TEST MATERIAL FORMULATIONS
Samples from each designated formulation, including the vehicle used to dose the control group, were analysed for the test material. During Week 6, individual achieved concentrations from the formulations prepared for use on Day 40 of the study (Groups 2 to 4) ranged from 113 to 126 % of the nominal. Since the majority of individual concentrations were above the acceptance criterion and contingency samples could not be analysed due to a lack of the frozen stability data, analysis of test material formulations prepared for use on Day 41 of the study (Groups 2 to 4 only) was performed.

METHOD OF ANALYSIS
The analytical procedure for the determination of the test material in water was conducted using UV/visible spectrophotometry using the test material, water and HPLC grade methanol.
- Instrument: Unicam UV/visible spectrophotometer

PROCEDURE
Linearity of Detection
The linearity of detection was assessed from triplicate measurements of six standards prepared at concentrations over the calibration range of the assay. The standard concentration and response data were subjected to linear regression analysis and the assay considered linear if the correlation coefficient (r) was no less than 0.99.
The linear correlation coefficient over the calibration range 20.32 to 81.80 µg/mL of the test material was 0.9968.

Precision of Detection
The precision of detection was assessed over the linear range from the coefficient of variation of the response factors (response/concentration) of all standards analysed in triplicate.
The precision of detection at the mid-point of the linear range was assessed from the coefficient of variation of the responses of a mid-point standard analysed six times consecutively. The precision of detection was considered acceptable if the coefficients of variation were no greater than 7.5 % for each assessment.
The precision of detection over the linear range was 3.4 %. The precision of detection at the mid-point of the linear range was 0 %.

System Stability
The stability of the system was assessed from the coefficient of variation of the responses of a mid-point standard, analysed six times interspersed throughout the run, and was considered acceptable if no greater than 7.5 %.
The stability of the system was 0.1 %.

Specificity
The specificity of the assay was assessed from the analysis of control samples and reagent blanks and was considered acceptable if the absorbance was no greater than 5 % of that of the lowest concentration standard.
No response that was attributed to the test material was observed in the control samples or reagent blanks.

Intra-Assay Accuracy and Precision
The intra-assay accuracy at each concentration, expressed as the mean accuracy values (n=3) was assessed from the analysis of triplicate samples of the test material in water, at concentrations of 10 mg/mL and 200 mg/mL. The assay was considered accurate if the individual and the mean measured concentrations of the test material were between 85 and 115 % of their nominal values. The intra-assay precision, expressed as the coefficient of variation of the individual accuracy values at each concentration, was considered acceptable if no greater than 10 %.
The individual accuracy values at 10 mg/mL were between 85 and 89 % with a mean value of 87 % and a precision value of 2.5 %. The individual accuracy values at 200 mg/mL were between 85 and 87 % with a mean value of 86 % and a precision value of 1.1 %.

Inter-Assay Accuracy and Precision
Assay accuracy and precision, were repeated on a separate occasion, using freshly prepared solutions and reagents, to establish the effects of such changes.
The inter-assay accuracy at each concentration, expressed as the mean accuracy values (n=6) obtained over two occasions, was considered acceptable if the individual and the mean measured concentrations of the test material were between 85 and 115 % of their nominal values.
The inter-assay precision at each concentration, expressed as the coefficient of variation of the individual accuracy values (n=6) obtained over two occasions, was considered acceptable if no greater than 15 %.
The individual accuracy values at 10 mg/mL were between 85 and 95 % with a mean value of 90 % and a precision value of 4.3 %. The individual accuracy values at 200 mg/mL were between 85 and 89 % with a mean value of 88 % and a precision value of 2.0 %.

RESULTS OF FORMULATION ANALYSIS
Formulations used to dose animals during Week 1 of the study were considered accurate since the measured concentrations were within 15 % of their nominal values which fulfilled the acceptance criterion.
The majority of samples from test formulations used to dose animals in Groups 2, 3 and 4 on Day 40 of the study (Week 6) showed higher levels of the test material than the target values (by up to 26 %), which was outside the specified acceptance criterion. Test material formulations used to dose animals in Groups 2, 3 and 4 on Day 41 of dosing were also higher than their nominal values (by up to 30 %), which was again outside the specified acceptance criterion. Although no reason could be established for these higher values, considering these animals were dosed at higher dose levels than their target, this was considered to have no impact on the validity of the No Observed Adverse Effect Levels (NOAELs) determined in this study.
No test material was detected in vehicle used to dose animals in Group 1.

Duration of treatment / exposure:

The males were dosed for 14 days prior to, and during pairing, and until the day prior to necropsy. The females were dosed once daily for 14 days prior to, and during pairing, gestation and until Day 3 of lactation, inclusive.
Any female in mid-parturition at the time of dosing was not dosed on that day.

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of results from a preliminary study. A high dose level of 1000 mg/kg/day was chosen, as this is the maximum dose level required by the guidelines. At this dose level in the preliminary study, clinical signs remained confined to excessive pre and/or post-dose salivation.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity. From the start of treatment, all animals were examined daily for clinical signs of toxicity or changes in behaviour and appearance.
During the treatment period, each main study animal was routinely checked pre-dose and soon after completion of dosing. On week days, additional observations were made approximately 1 hour after dosing and either approximately 4 hours after dosing or at the end of the working day (whichever was sooner). At weekends and public holidays, additional observations were made approximately 1 hour after dosing or at the end of the working day (whichever was sooner).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed clinical examination once each week, from the start of treatment. Standard arena observations (functional observations) were made cage side and outside the home cage, in a standard arena, for all animals. Observations were recorded once prior to the start of dosing and once weekly thereafter during the dosing period, at approximately the same time of day on each occasion (afternoon). On Day 40 of the study, these observations were not recorded for any females in mid-parturition or on Day 0 of lactation; they were instead recorded on Day 42 of the study for those females.

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded at the start of treatment and then weekly thereafter up to and including the day of necropsy. Female body weights were recorded at the start of treatment and then at weekly intervals until the day of mating. Body weights for females were also recorded on Days 0, 7, 14 and 20 of gestation and then on Days 0 (where required for dose volume calculation), 1 and 4 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule: The amount of food consumed by the animals in each cage was recorded at weekly intervals for males and females during their pre-pairing dosing period. Food consumption of the females was also recorded over Days 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation and over Days 1 to 4 of lactation. Two weeks after the start of the pairing period, food consumption for males was recorded weekly up to and including the day prior to necropsy.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The animals were bled on Day 14 of dosing. Blood samples were taken from the sublingual vein (blood sample volume and anticoagulant: 0.5 mL EDTA; 0.5 mL 3.2 % w/v aqueous trisodium citrate; 0.5 mL lithium heparin).
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: No data
- How many animals: The first 5 animals/sex/group with the lowest identification numbers.
- Parameters examined: haemoglobin concentration, platelet count, red blood cell count, total leucocyte count, packed cell volume, leucocyte differential count, mean cell volume, reticulocytes, mean cell haemoglobin, cell morphology (blood smears were prepared) and mean cell haemoglobin concentration.
All parameters were measured on the ADVIA 120, haematology analyser.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The animals were bled on Day 14 of dosing. Blood samples were taken from the sublingual vein (blood sample volume and anticoagulant: 0.5 mL EDTA; 0.5 mL 3.2 % w/v aqueous trisodium citrate; 0.5 mL lithium heparin).
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: No data
- How many animals: The first 5 animals/sex/group with the lowest identification numbers.
- Parameters examined: urea, globulin (calculated), creatinine, A/G ratio, glucose, total bilirubin, alkaline phosphatase, total cholesterol, alanine aminotransferase, calcium, aspartate aminotransferase, sodium, total protein, potassium, albumin and bile acids.
Measured on the Roche Modular Evo (P800) clinical chemistry analyser.
All analyses except sodium and potassium were carried out at 37 °C. The analyses of sodium and potassium were carried out at 35 °C.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes - functional observation battery
Five animals of each sex in all groups were observed for sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity. For males these observations were recorded on Day 54 of the study and for females it was performed on Days 40 or 42 of the study (Days 1 to 3 of lactation).

OTHER: Coagulation parameters: prothrombin time and activated partial thromboplastin time. Measured on the ACL 9000.

Sacrifice and pathology:

GROSS PATHOLOGY
At the end of the treatment period, all animals were killed by exposure to carbon dioxide gas in a rising concentration. Females were killed on Day 4 of lactation. Males were killed after the females had been subjected to necropsy. All animals were weighed and examined externally. The abdominal cavity was opened and the animals exsanguinated from the caudal vena cava. The cranial and thoracic cavities were opened and a full internal examination was performed. Any macroscopic abnormalities were recorded and retained. The number of implantation scars and the number of corpora lutea for each female was recorded.

HISTOPATHOLOGY
The following tissues were removed and fixed as appropriate from all animals: epididymides*, animal identification, ovaries, prostate and seminal vesicles (incl. coagulating gland), testes*, uterus (incl. uterine cervix and oviducts), vagina and all gross lesions.

The following tissues were removed and fixed as appropriate from an additional 5 male and 5 female animals in each group: adrenal glands*, bone marrow smear, brain* (3 levels examined), caecum, colon, duodenum, femur and joint (incl. marrow), heart*, ileum (incl. Peyer’s patch), jejunum, kidneys*, liver*, lungs (incl. mainstem bronchi), mesenteric lymph node, sciatic nerve, spinal cord (3 levels examined), spleen*, stomach, submandibular lymph nodes, thymus*, thyroids (incl. parathyroids), trachea and urinary bladder.

The organs specified* in the tissue lists were weighed after trimming of fat and other contiguous tissue. Contralateral organs were weighed together.

FIXATIVES
With the exception of the bone marrow smear, testes and epididymides, either whole organs or samples of the tissues listed (where applicable) were preserved in neutral buffered formaldehyde. The testes and epididymides were fixed in Bouin’s fixative for 24 hours and then transferred to neutral buffered formaldehyde. The bone marrow smear was fixed in methanol for approximately 15 minutes.

TISSUES PROCESSED AND EXAMINED BY LIGHT MICROSCOPY
For control and high dose animals killed at the end of the treatment period, the tissues were wax embedded, cut at a nominal thickness of 4 to 5 µm, stained with haematoxylin and eosin and examined microscopically.
Following the initial microscopic examination, the following tissues from both males and females from the low and intermediate dose animals were wax embedded, cut at a nominal thickness of 4 to 5 µm, stained with haematoxylin and eosin and examined microscopically: femur and joint (incl. marrow) and mesenteric lymph nodes.

BONE MARROW SMEARS
Bone marrow smears were prepared and stained for all animals. The slides were suitable for a full myelogram assessment and those from control and high dose animals were examined.

Other examinations:

Further parameters examined in the parental generation as part of the screening for reproductive and developmental effects were: pairing, detection of mating and oestrous cycles, parturition and observations of littering females. Reproductive indices were also calculated.
Observations in the F1 generation included: litter size and sex, clinical observations, mortality and bodyweights. Offspring viability indices were calculated.

Statistics:

Non-parametric methods were employed if it was considered that the assumptions required for parametric analysis did not hold.
No analyses were performed for variables for which there were less than five independent observations in the control group; when treated groups had fewer than five observations analyses were performed at the discretion of the statistician.
Data from each sex was analysed separately. Statistical significance was declared at the p<0.05 level in all cases and noted at the p<0.01 and p<0.001 levels.

COMPARISONS
Group 1 (control) against Groups 2, 3 and 4 (100, 300 and 1000 mg/kg, respectively)

STATISTICAL TESTS AND PARAMETERS
Data were processed to give group mean values and standard deviations where appropriate. Where the data allowed, the following methods were used for statistical analysis:

ANALYSIS OF VARIANCE AND WILLIAMS' TEST
(Non-parametric alternative: Kruskal-Wallis ANOVA and Shirley's Test)
Body weights and body weight gains, Food consumption, Organ weights (absolute), Relative organ weights (organ weights as a percentage of terminal body weight), Clinical pathology data, Grip strength and Motor activity (timepoints 1 and 1 - 6 (total)).

ANALYSIS OF COVARIANCE
Motor activity (timepoints 2 to 6)

ANALYSIS OF VARIANCE
(Non-parametric alternative: Kruskal-Wallis Test)
Pre-dose body weights (Day 1)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

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

Description (incidence and severity):

see below

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see below

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):

see below

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL OBSERVATIONS
There were no clinical signs considered to be related to the toxicity of the test material.
From Day 7 of the study, clinical observations for males and females given 1000 mg/kg/day included excessive salivation predominantly seen at pre-dose and/or immediately after dosing and persisted sporadically throughout the dosing period. At 300 mg/kg/day, sporadic incidences of pre- and/or post-dose salivation were noted in males from Day 28 of the study and some females given the same dose level also showed signs of excessive salivation immediately after dosing on Days 31 and 32 of the study. These observations were considered to be related to the dosing procedure and/or taste of the test material formulations.
Throughout the treatment period, sporadic incidences of agitation and vocalisation were noted for individual females from all groups receiving the test material. These were also noted for some control females during the recording of functional observations, and therefore, considered not to be related to the toxicity of the test material.

BODYWEIGHTS
Summary data is included in Tables 1 and 2.
During the pre-pairing period, group mean bodyweight gains for males given 1000 mg/kg/day were slightly lower than controls, achieving statistical significance during the first week of dosing (p<0.05). For the remainder of the treatment period, group mean bodyweight gains for these males were generally similar to controls. Overall mean bodyweight gain for this group of animals was slightly but statistically significantly lower (p<0.05) in comparison with controls. For males given 100 or 300 mg/kg/day, mean bodyweight gains were generally similar to controls throughout the treatment.
During the first week of the pre-pairing period, females given 1000 mg/kg/day showed a stasis in group mean bodyweight with half of these animals losing body weight; in comparison control animals gained weight during this period and the difference was statistically significant (p<0.001). Thereafter, an improvement was evident such that during the second week of dosing, the mean bodyweight gain for these females was higher than controls, albeit without achieving statistical significance. Group mean bodyweight gains for these females during the gestation and lactation periods remained unaffected. For females receiving 100 or 300 mg/kg/day of the test material, there was no effect of treatment on body weight gain during any stage of the study.

FOOD CONSUMPTION
At 1000 mg/kg/day, food consumption for males was generally similar to controls throughout the dosing period. Females given the same dose level showed slightly lower food intake during the pre-pairing period and over Days 0 to 4 of gestation (p<0.05) when compared with controls. Throughout the remainder of the gestation period, the mean food intake values for this group of females were marginally lower than controls, albeit without achieving statistical significance. During lactation, food intake values for these females were similar to controls.
For animals given 100 or 300 mg/kg/day of the test material, food intake values were generally similar to controls throughout the study.

HAEMATOLOGY AND COAGULATION
Selected parameters are included in Table 3.
At all dose levels, males showed a statistically significant, albeit non dose-related, lowering of total leucocyte counts (p<0.05) with respect to controls, due to lower absolute lymphocyte counts. There was no clear dose-relationship and the individual values were within the background control data ranges but, in view of histopathology findings in the bone marrow of males given 1000 mg/kg/day, a relationship with treatment cannot be entirely excluded. Corresponding leucocyte and lymphocyte values in all female groups given the test material were also slightly lower than the respective control group, albeit without achieving statistical significance. In the absence of any associated histopathology findings, this was considered not to be of toxicological significance.
At 1000 mg/kg/day, females showed a marginal but statistically significant lowering in haemoglobin concentration (p<0.05) and slight prolongation of prothrombin time (p<0.001) when compared with controls. Individual haemoglobin concentrations were within the background control data range but respective prothrombin times were above these ranges. In the absence of any associated histopathology changes these findings were considered not to be toxicologically significant.

BLOOD CHEMISTRY
Statistically significantly higher mean plasma concentration of bile acids (p<0.05) was observed in females given 1000 mg/kg/day with respect to controls. However, only one out of 5 males at this dose level had higher bile acid concentration than controls. At 100 or 300 mg/kg/day, a small number of males and females also showed higher concentrations of bile acids than controls. High individual variation seen in these animals may be due to the fact that these animals were not fasted prior to bleeding and the intergroup differences were considered unrelated to treatment.
Statistically significant, higher plasma glucose concentrations (p<0.01) were observed in males given the test material at 300 or 1000 mg/kg/day, albeit without any dose-relationship. These males also showed a statistically significant lowering in plasma levels of sodium (p<0.001) whereas statistically significantly higher levels of cholesterol (p>0.01) were seen in males given 1000 mg/kg/day. The majority of individual values were, however, within the background data ranges and were considered not to be of toxicological significance.

FUNCTIONAL OBSERVATIONS
There were no clear effects of treatment on functional observations monitored throughout the study or sensory reactivity to visual, acoustic, tactile or proprioceptive stimuli monitored towards the end of the study.
- Grip Strength: Any differences between control and treated animals of either sex were non dose-related and individual variation was high amongst animals. These differences were considered not related to treatment with the test material.
- Motor Activity: Any differences between control and treated animals were non dose-related and individual variation was high amongst animals. These differences were considered not related to treatment with the test material.

ORGAN WEIGHTS
At 1000 mg/kg/day, absolute and bodyweight-related liver weights for animals of both sexes were statistically significantly higher than controls (p<0.001 for males and p<0.01 for females). Individual bodyweight-related liver weights for males were within the background control data ranges whereas for females, including controls, the majority of corresponding individual values were above the background control data ranges. In the absence of any histopathology findings for the liver, the increases in liver weight were considered to be adaptive in nature and not indicative of toxicity.
At 300 or 1000 mg/kg/day, females also showed statistically significantly lower body weight-related heart weights than controls (p<0.05) but without any dose-relationship. The majority of individual values were within the background control data ranges and as there were no corresponding histopathology findings, this finding was considered not to be related to treatment with the test material.

MICROSCOPIC FINDINGS
Findings considered to be related to treatment were recorded in the bone marrow of the males (increase in marrow adipocytes) given the test material at a dose level of 1000 mg/kg/day. There was an equivocal change (erythrocytosis/erythrophagia) in the mesenteric lymph nodes for animals of both sexes receiving 1000 mg/kg/day. No effect was noted for these parameters in the low or intermediate dose groups. In addition, an assessment of the bone marrow smears from control animals and those given 1000 mg/kg/day indicated that there was no effect of treatment on the assessed parameters. The pertinent findings are summarised in Table 4.
A small erosion was noted in the non-glandular stomach of one male given 1000 mg/kg/day of the test material. Associated with this were epithelial hyperplasia, inflammation and oedema. Small lesions of this nature in the fore stomach are not uncommon with gavage studies and it is not thought to be indicative of a local irritant effect of the formulation nor of any systemic effect. A variety of other spontaneous changes was noted in control and treated animals with no indication of an effect of treatment. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.

Effect levels

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1 Bodyweights and Bodyweight Gains (g) for P Generation Males - Group Mean Values

Dose (mg/kg/day)		Day Number
		1#	8	15	22	29	36	43	50	55#
0	Mean SD N	303.9 9.8 10	322.4 11.2 10	338.6 13.3 10	346.8 13.6 10	363.9 15.4 10	373.4 18.3 10	385.0 16.7 10	397.1 19.1 10	399.5 20.1 10
100	Mean SD N	297.0 10.0 10	312.8 9.2 10	330.2 14.0 10	343.3 15.0 10	360.3 14.1 10	370.4 16.6 10	378.1 19.2 10	389.0 20.6 10	391.4 19.4 10
300	Mean SD N	311.0 12.3 10	328.1 17.8 10	344.7 19.4 10	357.0 19.9 10	369.5 24.9 10	382.4 26.8 10	391.6 26.4 10	401.8 23.7 10	405.7 25.4 10
1000	Mean SD N	304.8 13.7 10	317.5 13.6 10	330.6 13.0 10	340.9 14.6 10	352.2 15.8 10	360.6 16.3 10	371.4 17.9 10	379.2 21.7 10	385.3 21.6 10

 

Dose (mg/kg/day)		Day Number
		Gain# 1 to 8	Gain# 8 to 15	Gain# 15 to 22	Gain# 22 to 29	Gain# 29 to 36	Gain# 36 to 43	Gain# 43 to 50	Gain# 50 to 55	Gain# 1 to 55
0	Mean SD N	18.5 5.3 10	16.2 5.1 10	8.2 3.3 10	17.1 3.6 10	9.5 7.6 10	11.6 5.3 10	12.1 3.9 10	2.4 3.4 10	95.6 15.7 10
100	Mean SD N	15.8 3.6 10	17.4 5.9 10	13.1 5.2 10	17.0 4.7 10	10.1 3.1 10	7.7 5.2 10	10.9 4.5 10	2.4 3.7 10	94.4 12.5 10
300	Mean SD N	17.1 7.5 10	16.6 2.8 10	12.3 4.8 10	12.5* 8.1 10	12.9 4.2 10	9.2 5.7 10	10.2 5.5 10	3.9 4.8 10	94.7 15.1 10
1000	Mean SD N	12.7* 4.4 10	13.1 3.5 10	10.3 6.2 10	11.3* 5.0 10	8.4 3.8 10	10.8 3.0 10	7.8 5.7 10	6.1* 2.0 10	80.5* 18.3 10

# = Statistically analysed

SD = Standard deviation

N = Number of animals

* = p<0.05

 

Table 2 Bodyweights and Bodyweight Gains (g) for P Generation Females - Group Mean Values

Dose (mg/kg/day)		Day Pre-pairing
		1#	8	15#	Gain# 1 to 8	Gain# 8 to 15	Gain# 1 to 15
0	Mean SD N	201.7 8.1 10	210.5 9.3 10	215.4 9.5 10	8.8 4.4 10	4.9 3.9 10	13.7 6.5 10
100	Mean SD N	195.9 6.7 10	203.8 10.7 10	211.7 10.4 10	7.9 5.8 10	7.9 7.1 10	15.8 6.5 10
300	Mean SD N	207.2 7.6 10	212.2 7.7 10	218.9 9.5 10	5.0 5.0 10	6.7 3.9 10	11.7 4.6 10
1000	Mean SD N	202.0 7.1 10	202.2 7.2 10	211.4 7.2 10	0.0*** 4.6 10	9.4 5.7 10	9.4 5.6 10

 

Dose (mg/kg/day)		Day Pre-pairing								Day of Lactation
		0#	7	14#	20	Gain# 0 to 7	Gain# 7 to 14	Gain# 14 to 20	Gain# 0 to 20	1#	4#	Gain# 1 to 4
0	Mean SD N	221.4 16.3 10	245.6 16.9 10	274.4 18.1 10	335.6 19.4 10	24.2 4.9 10	28.8 4.2 10	61.2 9.8 10	114.2 10.4 10	258.5 16.3 10	271.9 17.8 10	13.4 7.5 10
100	Mean SD N	209.6 11.4 10	234.9 17.2 10	261.9 24.0 10	322.5 31.2 10	25.3 9.8 10	27.0 8.1 10	60.6 9.3 10	112.9 23.5 10	248.6 22.1 10	262.0 20.4 10	13.4 8.6 10
300	Mean SD N	220.6 10.1 10	244.0 11.0 10	271.8 15.2 10	336.1 15.4 10	23.4 8.2 10	27.8 5.6 10	64.3 7.5 10	115.5 13.2 10	260.8 11.2 10	270.0 11.4 10	9.2 5.7 10
1000	Mean SD N	212.0 6.6 10	234.8 10.5 10	262.9 13.0 10	324.6 20.5 10	22.8 7.3 10	28.1 5.5 10	61.7 11.4 10	112.6 15.0 10	253.9 11.1 10	267.5 8.1 10	13.6 8.3 10

# = Statistically analysed

SD = Standard deviation

N = Number of animals

* = p<0.001

 

Table 3 Selected Haematology and Coagulation Parameters, P Generation Day 14 - Group Mean Values

Sex	Dose (mg/kg/day)		Hb# (g/dL)	WBC# (10³/μL)	Lymph# (10³/μL)	PT+# (seconds)
Male	0	Mean SD N	15.4 0.6 5	9.20 0.71 5	7.52 0.58 5	21.0 1.1 5
	100	Mean SD N	15.6 0.3 5	7.57* 1.12 5	6.08** 0.82 5	20.3 1.1 5
	300	Mean SD N	15.2 0.4 5	7.18* 1.18 5	5.55** 0.59 5	21.2 0.7 5
	1000	Mean SD N	15.2 0.8 5	7.34* 1.22 5	6.10** 0.96 5	20.7 1.0 5
Female	0	Mean SD N	14.1 0.2 5	6.15 0.91 5	4.84 0.52 5	21.3 1.2 5
	100	Mean SD N	14.0 0.3 5	5.35 0.84 5	4.27 0.63 5	21.6 0.6 5
	300	Mean SD N	14.0 0.2 5	5.61 1.33 5	4.06 0.79 5	20.8 0.8 5
	1000	Mean SD N	13.5* 0.6 5	5.15 1.50 5	4.19 1.19 5	23.9*** 0.8 5

# = Statistically analysed

SD = Standard deviation

N = Number of animals

* = p<0.05

** = p<0.01

*** = P<0.001

 

Table 4 Summary of Pertinent Microscopic Findings

	Males				Females
Number Examined	5	5	5	5	5	5	5	5
Dose (mg/kg/day)	0	100	300	1000	0	100	300	1000
Bone Marrow Marrow adipocytes
Minimal	0	0	2	0	4	5	4	3
Slight	5	3	3	0	0	0	1	0
Moderate	0	2	0	4	0	0	0	0
Marked	0	0	0	1	0	0	0	0
Total	5	5	5	5	4	5	5	3
Mesenteric Lymph Nodes Erythrocytosis/erythrophagia
Minimal	1	2	2	4	2	1	2	2
Slight	0	0	0	1	0	1	0	2
Total	1	2	2	5	2	2	2	4

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, in the Wistar strain rat, the No Observed Adverse Effect Level (NOAEL) for male and female repeated-dose toxicology was considered to be 300 mg/kg/day.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422 under GLP conditions.

Three groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (UHP water) following the same dosing regimen as the treated animals and were used as controls. The males were dosed for 14 days prior to pairing, during pairing and until the day prior to necropsy. The females were dosed for 14 days prior to pairing, during pairing and gestation, and up to and including Day 3 of lactation.

Clinical signs, body weights, food consumption and functional observations including grip strength and motor activity were recorded at pre-determined intervals. On Day 14 of dosing, blood samples were taken from 5 parental animals of each sex in all groups for clinical pathology evaluations. All animals were subjected to necropsy and tissues from control and high dose groups were microscopically examined.

There were no unscheduled deaths on the study and administration of the test material was well tolerated. Animals of both sexes given 1000 mg/kg/day showed slightly lower bodyweight gains than controls during the early part of the dosing period. Males given the test material at all dose levels had lower lymphocyte counts than controls and an increase in bone marrow adipocytes was seen in males at the highest dose level only. These findings were considered to be possibly treatment-related but the magnitude of change was small.

Under the conditions of this study, in the Wistar strain rat, the No Observed Adverse Effect Level (NOAEL) for male and female repeated-dose toxicology was considered to be 300 mg/kg/day.