Phenol, C14-18-alkyl derivs.

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 September 2014 to 27 October 2014

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 Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

C14-16-18 Alkyl phenol

EC Number:

931-468-2

Cas Number:

1190625-94-5

Molecular formula:

For substance information - see Section 1.2

IUPAC Name:

C14-16-18 Alkyl phenol

Test material form:

other: slightly viscous liquid

Details on test material:

- Appearance: Clear amber liquid
- Storage conditions of test material: At room temperature in the dark

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) (outbred, SPF-Quality)
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation (mean): Males: 144 g; Females: 124 g
- Fasting period before study: No, though during motor activity measurements animals had no access to food.
- Housing: Group housing of up to 5 animals per sex in cages (height 18 cm) with sterilised sawdust as bedding material and paper as cage-enrichment. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: ad libitum pelleted rodent diet
- Water: Free access to tap water except during motor activity measurements
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: At least 10 per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
From: No data
To: 27 October 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were stirred during dosing of all animals. Adjustment was made for specific gravity of the vehicle.

DOSE VOLUME: 5 mL/kg bw

VEHICLE
- Specific gravity: 1.036

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CHEMICAL ANALYSIS OF DOSE PREPARATIONS
Samples of dose preparations were taken on a single occasion during the treatment phase and were stored and dispatched on dry ice to the test site for formulation analysis.
Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90 to 110 % of target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.
Additionally, the long-term stability of the formulation samples prepared in advance of the study and stored at a target temperature =-70 °C was determined.

SAMPLES
In total, 16 samples were included in this study, distributed over 4 formulation groups. The samples of the control group and the 100 mg/kg dose group were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment groups 30 and 300 mg/kg were taken in duplicate from the top, middle and bottom position of the container.
The samples were stored at a target temperature =-70 °C.

ANALYTICAL METHOD
- Analytical conditions
The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
- Sample injections
The analytical run for the determination of the accuracy of preparation and homogeneity of the test material in formulations was acquired in the following order: analytical blank sample, calibration curve, analytical blank sample, analytical blank sample, procedural recovery sample high and low (replicate 1), analytical samples, long term storage stability samples and procedural recovery samples high and low (replicate 2).
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

FORMULAS
Response (Y): Peak area test material [mAU]

Calibration curve: Y = a + bX + cX²
where:
X = nominal concentration [mg/L]
c = curvature
b = slope
a = intercept

Analysed concentration (X): X = [-b + v(b² - 4c(a – y)) / 2c] x ((V x d) / w) [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
[-b + v(b² - 4c(a – y)) / 2c] values are obtained from Analyst® version 1.5.2 (Applied Biosystems, Burlington, Canada).

Recovery: (Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]

Accuracy: (Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]

Relative difference (relative diff.): ((Ct - C0) / C0) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

SPECIFICATIONS
- Run Acceptance
Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85 to 115 % were accepted. Outliers could be discarded as long as minimally 75 % of the responses of the calibration were accepted.
The mean recovery of the procedural recovery samples at each level had to be in the range 90 to 110 %.
The response in all analytical blank samples at retention time of the test material should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).
- Acceptance criteria formulations
Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 to 110 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.

RESULTS
- Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Regression analysis was performed using the least squares quadratic regression with a (1 / concentration²) weighting factor. The coefficient of correlation (r) was always higher than or equal to 0.9990.
No response at the retention time of the test material in the analytical blank samples was detected.
- Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 90 to 110 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.
- Test samples
In the control group formulations, no test material was detected. The concentrations analysed in the formulations of the dose groups were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %).
The formulations of the 30 and 300 mg/kg dose groups were homogeneous (i.e. coefficient of variation = 10 %).
- Stability at storage conditions
The mean accuracies were 98.7 and 101.0 % for the high (200 mg/g) and the low (1.00 mg/g) concentration levels, respectively, which are within the set criteria (mean accuracy is in the range 90 to 110 %). It was therefore concluded that the test material was stable in propylene glycol over a storage period of at least 40 days at a temperature of =-70 °C.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 100 and 300 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected on the basis of a 14 day range-finding study in which the rats were dosed at 500 and 1000 mg/kg bw.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality and viability were made at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from the start of treatment onwards, detailed clinical observations were made in all animals immediately after dosing (no peak effect of occurrence of clinical signs was observed in the dose range finding study). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected on the day of necropsy at the end of the treatment phase. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL) and with citrate for clotting tests (0.45 mL).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: All animals
- Haematology parameters examined: White blood cells (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils and basophils), red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and platelets.
- Clotting parameters examined: Prothrombin time (PT) and activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected on the day of necropsy at the end of the treatment phase. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: All animals
- Parameters examined: Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium and inorganic phosphate (Inorg. Phos).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 4 of treatment, the following tests were performed on all animals after dosing at no specific time point (but within a similar time period after dosing for all animals).
- Dose groups that were examined: All
- Battery of functions tested: hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R), fore- and hind-limb grip strength (recorded as the mean of three measurements, and locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerised monitoring system).

Sacrifice and pathology:

SACRIFICE
On the scheduled day of necropsy, animals were deeply anaesthetised using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied.

PATHOLOGY
Descriptions of all macroscopic abnormalities were recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10 % buffered formalin (neutral phosphate buffered 4 % formaldehyde solution): Adrenal glands, (aorta), brain [cerebellum, mid-brain, cortex], caecum, cervix, (clitoral gland), colon, duodenum, epididymides*, eyes (including optic nerve [if detectable] and harderian gland)*, (female mammary gland area), femur including joint, heart, ileum, jejunum, kidneys, (larynx), (lacrimal gland, exorbital), liver, lung infused with formalin, lymph nodes - mandibular, mesenteric, (nasopharynx), (oesophagus) , ovaries, (pancreas), peyer's patches [jejunum, ileum] if detectable, (pituitary gland), (preputial gland), prostate gland, rectum, (salivary glands - mandibular, sublingual), sciatic nerve, seminal vesicles including coagulating gland, skeletal muscle, (skin), spinal cord [cervical, midthoracic, lumbar], spleen, sternum with bone marrow, stomach, testes*, thymus, thyroid including parathyroid [if detectable], (tongue), trachea, urinary bladder, uterus, vagina and all gross lesions.
Tissues marked with * were fixed in modified Davidson's solution; tissues were transferred to formalin after fixation for at least 24 hours.
Tissues/organs mentioned in parentheses were not examined by the pathologist.

The following organ weights and terminal body weight were recorded from the animals on the scheduled day of necropsy: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus (including cervix), prostate*, seminal vesicles including coagulating glands* and thyroid including parathyroid.
Organs marked with * were weighed when fixed for at least 24 hours.

HISTOTECHNOLOGY
All organ and tissue samples were processed, embedded in paraffin wax, cut at a thickness of 2 to 4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY
The following slides were examined:
- All tissues collected at the scheduled sacrifice from all control and high dose animals;
- Stomach and liver (males and females) and kidneys (males) of low and mid-dose animals based on (possible) treatment-related changes in these organs in the high dose group; and
- All gross lesions.

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 if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
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. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Mortality:

mortality observed, treatment-related

Description (incidence):

see below

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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:

MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
Slight to moderate salivation was noted directly after dosing at 300 mg/kg/day starting from Week 1 to 2 onwards. In general the incidence and severity increased over time. From Week 2 onwards all animals were affected.
In addition, occasional slight salivation above background incidence was also noted for females treated at 100 mg/kg/day, with the highest incidence occurring in Week 4.
No further treatment related clinical signs were noted during detailed daily observations and weekly arena observations.
In males treated up to 100 mg/kg/day and females treated up to 30 mg/kg/day, salivation after dosing was seen at a low incidence and degree of severity. Salivation at this incidence and severity is occasionally seen in this strain of rats in this type of study and was considered to be unrelated to the test material.
Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions of this study and did not show any apparent dose-related trend.

BODY WEIGHTS
Body weights and body weight gain of treated animals remained in the same range as controls over the entire study period.

FOOD CONSUMPTION
Food consumption before or after correction for body weight remained similar to the control levels over the entire study period.

Haematology
The following statistically significant change in haematology parameters was noted:
- Higher white blood cell count in females at 300 mg/kg/day.
Other significant changes in haematological parameters occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain, and were therefore considered to be unrelated to treatment.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters were noted:
- Higher alanine aminotransferase activity (ALAT) in males and females 300 mg/kg/day;
- Higher alkaline phosphatase activity (ALP) in individual male and females at 300 mg/kg/day (not statistically significant);
- Lower total protein level in males at 300 mg/kg/day;
- Lower cholesterol level in males at 300 mg/kg/day;
- Higher potassium level in males at 300 mg/kg/day;
- Higher inorganic phosphate in males and females (not statistically significant).
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain.

NEUROBEHAVIOUR - FUNCTIONAL OBSERVATIONS
Higher forelimb grip strength was noted in females at 300 mg/kg/day at Week 4, with a slight dose-related trend.
Forelimb grip strength was considered normal in males and hind limb grip strength was considered normal in both sexes.
Hearing ability, pupillary reflex and static righting reflex were normal in all animals.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

ORGAN WEIGHTS
A higher relative and absolute liver weight was recorded for males (17.1 % higher mean relative liver weight compared to control) and females (higher mean absolute and relative liver weight compared to controls of 28.9 and 24.9 %, respectively) at 300 mg/kg/day. Relative liver weights were also higher in females at 30 and 100 mg/kg/day (8.2 and 8.5 %, respectively, compared to control).

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and/or had no treatment-related histopathological correlates. These necropsy findings were therefore considered to be unrelated to treatment with the test material.

MICROSCOPIC EXAMINATION
Test material-related findings in the forestomach were observed in 3/5 males of the 100 mg/kg/day group (2/5 females at 100 mg/kg regarded non-related to the test material) and in 5/5 males and 4/5 females of the 300 mg/kg/day group and consisted of:
- Lymphogranulocytic sub-epithelial inflammation in 3/5 males (2 minimal, 1 slight) and 2/5 females (1 minimal, 1slight) of the 300 mg/kg group.
- Squamous cell hyperplasia in 2/5 males (2 minimal) of the 100 mg/kg group and in 5/5 males (minimal) and 4/5 females (2 minimal, 2 slight) of the 300 mg/kg group.
- Increased incidences of hyperkeratosis in 3/5 males (3 minimal) of the 100 mg/kg/day group and 5/5 males (4 minimal, 1 slight) of the 300 mg/kg/day group, compared to 1/5 males (minimal) of the control group, 1/5 females (1 minimal) of the 30 mg/kg/day group, and 2/5 females (2 minimal) of the 100 mg/kg/day group.
- Erosion/ulceration in 2/5 males (2 minimal) and 1/5 females (1 minimal) of the 300 mg/kg/day group.
- Submucosal oedema in 2/5 males (2 minimal) of the 100 mg/kg/day and in 2/5 males (1 minimal, 1 slight) and 2/5 females (1 minimal, 1 slight) of the 300 mg/kg/day group.
Findings in the liver consisted of:
- Minimal hepatocellular hypertrophy in 2/5 males and 1/5 females at 30 mg/kg/day, in 1/5 males and 1/5 females at 100 mg/kg/day and in 3/5 males and 2/5 females at 300 mg/kg/day.
Findings in the male kidneys consisted of:
- Increased incidence and severity of hyaline droplet accumulation in 5/5 males at 300 mg/kg/day (3 minimal, 2 slight) compared to 1/5 control males (minimal), 2/5 males at 100 mg/kg/day (minimal) and 1/5 males at 30 mg/kg/day (minimal).
There were no other test material-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

DISCUSSION
The finding of higher forelimb grip strength recorded for females at 300 mg/kg/day was not supported by clinical observations or other functional observations, was slight in nature (well within the normal range for rats of this age and strain CI95 %:358 to 942), and had no supportive morphological correlates in examined neuronal tissues. In addition, a similar effect was not observed either for the hindlimb grip strength or in males. Therefore the higher grip strength was considered not toxicologically relevant.
Histopathological examination revealed minimal degree of erosion/ulceration in the forestomach of a few males and females at 300 mg/kg/day, along with hyperkeratosis, hyperplasia of the squamous epithelium, (sub)mucosal oedema, and/or sub-epithelial lymphogranulocytic inflammation (all at minimal to slight degree). Salivation was noted at this dose level at an incidence and severity above background level, which could be correlated to the irritancy of the test material, given as a bolus. In males at 100 mg/kg/day the forestomach lesions consisted of hyperkeratosis, hyperplasia of the squamous epithelium and/or (sub)mucosal oedema. These findings were considered to reflect damage of the forestomach epithelium by bolus gavage with the test material, primarily interruption of the protective forestomach epithelium by erosion/ulceration. Therefore these findings are regarded to be adverse in nature at the portal-of-entry.
The minimal degree of diffuse hyperkeratosis in a few females treated at 100 mg/kg/day was not accompanied by any additional forestomach lesion and can be noted at the same low degree in control rats as well. Occasional slight salivation above background incidence was noted in females at this dose level. These findings occurred at a low incidence and severity in the females of the 100 mg/kg/day group and are therefore considered not related to the test material.
Haematological examination revealed higher white blood cell count in females at 300 mg/kg/day. Although potentially related to treatment, this finding was considered not toxicologically relevant since clear morphological correlates in the stomach or individual white blood cell counts were absent, and the increase was only minor in nature.
Other changes in clinical biochemistry, such as higher potassium and inorganic phosphate occurred in the absence of clear morphological correlates or related changes in clinical laboratory parameters. The higher potassium and inorganic phosphate levels were within or just outside the normal range expected for this kind of rat and/or occurred in the absence of a clear dose response and was therefore considered not to be adverse.
Organ weight data showed higher absolute and/or relative liver weights in males and females at 300 mg/kg/day (maximally 25 % higher compared to controls), and in females at 30 and 100 mg/kg/day (both approximately 8 %). These changes in liver weight were considered to be related to a minimal degree of liver hepatocellular hypertrophy seen in both sexes at 30, 100 and 300 mg/kg/day and further accompanied by slight changes in clinical biochemistry parameters (i.e. higher alanine aminotransferase, lower total protein and cholesterol level) at the high dose level of 300 mg/kg/day only. Alkaline phosphatase activity was higher in some individual animals only. Although there was no morphological evidence of liver damage, the higher alanine aminotransferase activity and the increase in liver weight (maximally 25 %) are considered adverse in nature at 300 mg/kg/day. At 30 and 100 mg/kg/day the minimal increase in hypertrophy is not supported by toxicologically relevant liver weight or concurrent changes in clinical biochemistry parameters and hence is not considered to be adverse.
A treatment-related increase in the incidence and severity of hyaline droplet accumulation in male kidneys at 300 mg/kg/day was also noted at histopathology. This was considered not adverse in nature as it was likely due to accumulation of alpha2µglobulin, and was not accompanied by secondary proximal cortical tubule cell injury. Moreover, this male rat specific protein is not present in female rats or in higher mammals, including man.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

- local

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on morphologic changes in the forestomach, consisting of erosion/ulceration (and hyperkeratosis, hyperplasia of the squamous epithelium, (sub)mucosal oedema, and/or sub-epithelial lymphogranulocytic inflammation), a local NOAEL was established.

2

Dose descriptor:

NOAEL

Remarks:

- local

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on morphologic changes in the forestomach, consisting of erosion/ulceration (and hyperkeratosis, hyperplasia of the squamous epithelium, (sub)mucosal oedema, and/or sub-epithelial lymphogranulocytic inflammation), a local NOAEL was established.

3

Dose descriptor:

NOAEL

Remarks:

- systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the effects on liver, consisting of increases in weight and alanine aminotransferase activity, the systemic NOAEL was considered to be 100 mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of Selected Functional Observations

Grip Strength Parameter		Males				Females
		Dose Group (mg/kg)
		0	30	100	300	0	30	100	300
Fore (g)	Mean SD N	557 165 5	602 144 5	541 101 5	577 85 5	395 63 5	445 162 5	541 118 5	599* 113 5
Hind (g)	Mean SD N	500 44 5	440 75 5	490 135 5	546 79 5	344 79 5	387 122 5	402 44 5	397 58 5

*Significant at the 5 % level using the Dunnett-test based on pooled variance

 

Table 2: Selected Haematology Parameters

Parameter		Males				Females
		Dose Group (mg/kg)
		0	30	100	300	0	30	100	300
WBC (10E09/L)	Mean SD N	8.0 1.3 5	7.0 3.0 5	8.7 2.1 5	8.1 2.2 5	3.9 1.1 5	4.1 0.8 5	4.3 0.9 5	6.6** 1.1 5
MCH (fmol)	Mean SD N	1.22 0.03 5	1.15* 0.04 5	1.23 0.04 5	1.17 0.04 5	1.16 0.01 5	1.18 0.03 5	1.17 0.02 5	1.18 0.03 5
MCHC (mmol/L)	Mean SD N	21.81 0.29 5	20.85** 0.32 5	21.51 0.46 5	21.32 0.44 5	21.65 0.23 5	21.96 0.20 5	21.98 0.31 5	21.86 0.43 5

WBC = White blood cells

MCH = Mean corpuscular haemoglobin

MCHC = Mean corpuscular haemoglobin concentration

*Significant at the 5 % level using the Dunnett-test based on pooled variance

**Significant at the 1 % level using the Dunnett-test based on pooled variance

 

Table 3: Selected Clinical Chemistry Parameters

Parameter		Males				Females
		Dose Group (mg/kg)
		0	30	100	300	0	30	100	300
ALAT (U/L)	Mean SD N	46.3 4.5 5	55.9 9.5 5	62.8 4.4 5	122.8** 25.2 5	35.3 6.8 5	44.3 5.4 5	44.5 7.6 5	82.8** 36.5 5
ALP (U/L)	Mean SD N	268 91 5	263 65 5	294 72 5	446 247 5	130 62 5	125 43 5	193 64 5	221 180 5
Total protein (g/L)	Mean SD N	62.5 1.0 5	62.7 2.9 5	60.3 2.5 5	58.9* 1.2 5	62.7 1.8 5	64.5 2.3 5	64.7 1.7 5	60.7 2.4 5
Cholesterol (mmol/L)	Mean SD N	1.99 0.24 5	1.89 0.26 5	1.68 0.30 5	1.48* 0.23 5	1.36 0.32 5	1.29 0.29 5	1.34 0.21 5	1.29 0.21 5
Potassium (mmol/L)	Mean SD N	3.98 0.14 5	3.88 0.18 5	4.23 0.17 5	4.50** 0.32 5	3.50 0.27 5	3.68 0.48 5	3.68 0.26 5	3.82 0.27 5
Inorganic Phosphate (mmol/L)	Mean SD N	2.49 0.15 5	2.66 0.37 5	2.55 0.33 5	2.90 0.15 5	2.19 0.32 5	2.01 0.14 5	2.18 0.46 5	2.45 0.25 5

ALAT = Alanine aminotransferase

ALP = Alkaline phosphatase

*Significant at the 5 % level using the Dunnett-test based on pooled variance

**Significant at the 1 % level using the Dunnett-test based on pooled variance

 

Table 4: Selected Organ Weights and Organ Weight/Body Weight Ratios

Parameter		Males				Females
		Dose Group (mg/kg)
		0	30	100	300	0	30	100	300
Liver (g)	Mean SD N	8.07 0.84 5	9.00 0.63 5	8.49 0.66 5	9.32 1.00 5	4.71 0.61 5	5.25 0.24 5	5.44 0.50 5	6.07** 0.40 5
Liver/Body Weight Ratio (%)	Mean SD N	2.92 0.11 5	3.16 0.24 5	3.11 0.17 5	3.42** 0.19 5	2.81 0.08 5	3.04* 0.14 5	3.05* 0.08 5	3.51** 0.16 5

*Significant at the 5 % level using the Dunnett-test based on pooled variance

**Significant at the 1 % level using the Dunnett-test based on pooled variance

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study the local NOAEL was determined to be 30 and 100 mg/kg/day for males and females, respectively. The systemic NOAEL was determined to be 100 mg/kg/day for males and females.

Executive summary:

The repeated dose toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 407, EU Method B.7 and US EPA OPPTS 870.3050 under GLP conditions.

The test material, formulated in propylene glycol, was administered daily for 28 days by oral gavage to SPF-bred Wistar rats at dose levels of 0, 30, 100 and 300 mg/kg/day. Five rats per sex per dose were treated and chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters were evaluated: mortality, clinical signs, functional observations, body weight, food consumption, haematology and clinical chemistry. At the end of the treatment period the rats were sacrificed and subjected to a complete necropsy including gross pathology and histopathology examinations.

Formulation analysis confirmed that formulations of the test material in propylene glycol were prepared accurately and were homogenously distributed.

No mortality occurred and no changes in body weight and food consumption or findings at macroscopic examination were observed.

A finding of higher forelimb grip strength recorded for females at 300 mg/kg/day was not supported by any other findings and was considered to be not toxicologically relevant.

Morphologic changes were observed in the forestomach, consisting of erosion/ulceration (and hyperkeratosis, hyperplasia of the squamous epithelium, (sub)mucosal oedema, and/or subepithelial lymphogranulocytic inflammation) at 100 and 300 mg/kg/day for males and 300 mg/kg/day for females. These findings are regarded to be adverse in nature at the portal-of-entry.

Haematological examination revealed higher white blood cell count in females at 300 mg/kg/day, however this finding was considered not toxicologically relevant since clear morphological correlates in the stomach or individual white blood cell counts were absent, and the increase was only minor in nature. Other changes in clinical biochemistry, such as higher potassium and inorganic phosphate occurred in the absence of clear morphological correlates or related changes in clinical laboratory parameters. The higher potassium and inorganic phosphate levels were within or just outside the normal range expected for this kind of rat and/or occurred in absence of a clear dose response and therefore were considered not to be adverse.

Organ weight data showed higher absolute and/or relative liver weights in males and females at 300 mg/kg/day and in females at 30 and 100 mg/kg/day. These changes were considered to be related to a minimal degree of liver hepatocellular hypertrophy seen in both sexes at all dose levels and further accompanied by slight changes in clinical biochemistry parameters (i.e. higher alanine aminotransferase, lower total protein and cholesterol level) at the high dose level only. Alkaline phosphatase activity was higher in some individual animals only. Although there was no morphological evidence of liver damage, the higher alanine aminotransferase activity and the increase in liver weight are considered adverse in nature at 300 mg/kg/day. At 30 and 100 mg/kg/day the minimal increase in hypertrophy is not supported by toxicologically relevant liver weight or concurrent changes in clinical biochemistry parameters and hence is not considered to be adverse.

Under the conditions of this study the local NOAEL was determined to be 30 and 100 mg/kg/day for males and females, respectively. The systemic NOAEL was determined to be 100 mg/kg/day for males and females.