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Administrative data

Description of key information

28-Day gavage study; rat; NOAEL 100 mg/kg; Reliability = 1

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct. Additional histopathology was completed on stored tissues in 2005 to bring the study closer to the 1995 edition of OECD Guideline 407.
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
other: Crl:CD® (SD) BR VAF Plus
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 28 ± 1 days
- Weight at study initiation: 60-87 g
- Fasting period before study: not reported
- Housing: groups of 5 according to sex; cages distributed in batteries to minimize environmental influences from spatial distribution
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8-21.1, mean minimum and maximum temperatures
- Humidity (%): 55.0-59.9%, mean minimum and maximum humidities
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
-Preparation frequency: daily
-Preparation details: The test substance was homogenized with the vehicle to obtain a suspension
-Adjusted for purity: no

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported
- Concentration in vehicle: test substance concentration was 10.0, 1.0 and 0.1% w/v
- Amount of vehicle (if gavage): total dosage volume was 10 mL/kg/day
- Lot/batch no. (if required): not reported
- Purity: not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method of Analysis: HPLC

Concentration verification (% of Target)
Conducted on all dose levels: yes
Results:
Table 1: Mean concentration of test substance in dose formulations
Test Concentration Time Frame Result
1 mg/L Day 1, Replicate 1 0.97533 mg/mL
1 mg/L Day 1, Replicate 2 1.0398 mg/mL
10 mg/L Day 1, Replicate 1 10.919 mg/mL
10 mg/L Day 1, Replicate 2 10.167 mgm/L
100 mg/L Day 1, Replicate 1 108.17 mgmL
100 mg/L Day 1, Replicate 2 103.8 mg/mL
Control Day 1, Replicate 1 ND
Control Day 1, Replicate 2 ND

1 mg/L Day 22, Replicate 1 0.97465 mg/mL
1 mg/L Day 22, Replicate 2 1.0134 mg/mL
10 mg/L Day 22, Replicate 1 10.809 mg/mL
10 mg/L Day 22, Replicate 2 10.194 mg/mL
100 mg/L Day 22, Replicate 1 115.55 mg/mL
100 mg/L Day 22, Replicate 2 109.73 mg/mL
Control Day 22, Replicate 1 ND
Control Day 22, Replicate 2 ND

ND = Not Detected (limit of detection = 1.479x10e-4 mg/mL)
Homogeneity is reported in Table 2 below.
Conducted on dose levels: 1 and 100 mg/ml concentrations
Results:
Table 2: Physical stability of test substance in 1% methylcellulose formulations
Test Concentration Time Frame Result
1 mg/mL Top, Storage 0 hours 0.93169 mg/mL
1 mg/mL Middle, Storage 0 hours 1.0201 mg/mL
1 mg/mL Bottom, Storage 0 hours 0.95525 mg/mL
1 mg/mL Mean, Storage 0 hours 0.96901 mg/mL
1 mg/mL Top, Storage 0.5 hours 0.95106 mg/mL
1 mg/mL Middle, Storage 0.5 hours 0.94207 mg/mL
1 mg/mL Bottom, Storage 0.5 hours 0.93246 mg/mL
1 mg/mL Mean, Storage 0.5 hours 0.94186 mg/mL
1 mg/mL Top, Storage 1 hours 0.97522 mg/mL
1 mg/mL Middle, Storage 1 hours 0.98487 mg/mL
1 mg/mL Bottom, Storage 1 hours 0.84032 mg/mL
1 mg/mL Mean, Storage 1 hours 0.93347 mg/mL
1 mg/mL Top, Storage 4 hours 0.89724 mg/mL
1 mg/mL Middle, Storage 4 hours 0.88917 mg/mL
1 mg/mL Bottom, Storage 4 hours 0.93672 mg/mL
1 mg/mL Mean, Storage 4 hours 0.90771 mg/mL

100 mg/mL Top, Storage 0 hours 106.26 mg/mL
100 mg/mL Middle, Storage 0 hours 104.73 mg/mL
100 mg/mL Bottom, Storage 0 hours 104.49 mg/mL
100 mg/mL Mean, Storage 0 hours 105.16 mg/mL
100 mg/mL Top, Storage 0.5 hours 108.44 mg/mL
100 mg/mL Middle, Storage 0.5 hours 99.325 mg/mL
100 mg/mL Bottom, Storage 0.5 hours 103-57 mg/mL
100 mg/mL Mean, Storage 0.5 hours 103-78 mg/mL
100 mg/mL Top, Storage 1 hours 106.86 mg/mL
100 mg/mL Middle, Storage 1 hours 104.44mg/mL
100 mg/mL Bottom, Storage 1 hours 104.81 mg/mL
100 mg/mL Mean, Storage 1 hours 105.37 mg/mL
100 mg/mL Top, Storage 4 hours 107.88 mg/mL
100 mg/mL Middle, Storage 4 hours 108.94 mg/mL
100 mg/mL Bottom, Storage 4 hours 103.17 mg/mL
100 mg/mL Mean, Storage 4 hours 106.66 mg/mL

Stability (% of Time Zero)
Conducted on dose levels: 1 and 100 mg/mL concentrations
Storage was at room temperature in the dark
Results:
Table 3: Chemical stability of test substance in 1% methylcellulose formulations
Test Concentration Time Frame Result
1 mg/mL Storage 0 hours, Replicate 1 1.1313 mg/mL
1 mg/mL Storage 0 hours, Replicate 2 0.81800 mg/mL
1 mg/mL Storage 0 hours, Mean 0.97465 mg/mL
1 mg/mL Storage 4 hours, Replicate 1 0.93275 mg/mL
1 mg/mL Storage 4 hours, Replicate 2 0.90313 mg/mL
1 mg/mL Storage 4 hours, Mean 0.91794 mg/mL
1 mg/mL Storage 24 hours, Replicate 1 0.93222 mg/mL
1 mg/mL Storage 24 hours, Replicate 2 0.86380 mg/mL
1 mg/mL Storage 24 hours, Mean 0.89801 mg/mL

100 mg/mL Storage 0 hours, Replicate 1 102.50 mg/mL
100 mg/mL Storage 0 hours, Replicate 2 104.39 mg/mL
100 mg/mL Storage 0 hours, Mean 103.45 mg/mL
100 mg/mL Storage 4 hours, Replicate 1 102.71 mg/mL
100 mg/mL Storage 4 hours, Replicate 2 106.77 mg/mL
100 mg/mL Storage 4 hours, Mean 104.74 mg/mL
100 mg/mL Storage 24 hours, Replicate 1 106.20 mg/mL
100 mg/mL Storage 24 hours, Replicate 2 118.79 mg/mL
100 mg/mL Storage 24 hours, Mean 112.50 mg/mL
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control and high dose groups consisted of 10 animals/sex, for which 5 animals/sex were used in a recovery group. Low and middle dose groups consisted of 5 animals/sex.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on an acute oral toxicity study (HRC Report no. 90505D/HST 327/AC) and a preliminary toxicity study (HRC Report no. 90654D/HST 336/ST. In the preliminary study, rats were given seven daily gavage doses of 1000 mg/kg of the test substance suspended in 1% aqueous methylcellulose. No treatment-related effects were found.
- Rationale for animal assignment (if not random): animal assignment was random to minimize body weight variation between groups
- Rationale for selecting satellite groups: based on animal numbers
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity; daily for signs of ill health, behavioural changes or toxicosis.

BODY WEIGHT: Yes
- Time schedule for examinations: before dosing and subsequently at weekly intervals

FOOD CONSUMPTION :
- Food consumption for each cage determined weekly: Yes


WATER CONSUMPTION : Yes
- Time schedule for examinations: daily monitoring by visual appraisal of the water bottles

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 30 for main study groups; Day 44 for recovery groups
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked in Table 4 below were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 30 for main study groups; Day 44 for recovery groups
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked in Table 4 below were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Day 30 for main study groups; Day 44 for recovery groups
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked in Table 4 below were examined.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 5 below)
HISTOPATHOLOGY: Yes (see Table 5 below)

A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation and exsanguinated. Microscopic examination was performed on all tissues from all animals in the control and high-dose groups euthanized at the scheduled primary necropsy. Gross lesions were examined from animals in the low- and mid-dose groups euthanized at the primary necropsy and animals in the control and high-dose groups euthanized at the recovery necropsy. In addition, the liver was evaluated from animals in the low- and mid-dose groups euthanized at the primary necropsy, as well as animals in the control and high-dose group at the recovery necropsy.
Statistics:
Food consumption data were analysed on a cage basis using cumulative cage totals. The individual animal was the basic unit for all other parameters. Body weight data were analysed using weight gains. The following sequence of statistical tests were used for body weight, food consumption, organ weight and clinical pathology data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of values different from the mode were analysed by appropriate methods. Otherwise, Bartlett's test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used. Analyses of variance were followed by Student's 't' test and Williams' test for a dose-related response, although only the one thought more appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the 't' test and Williams' test (Shirley's test). Organ weight analysis initially involved a correlation analysis between organ weights and final body weight. For organs where a correlation at the 10% level of significance was established, analysis of organ weight data was performed using adjusted organ weights by analysis of covariance with final body weight as covariate. Where a correlation between organ weight and body weight was not established the organ weight analysis was carried out using routine analysis of variance on unadjusted organ weights.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Pilo-erection, often accompanied by increased salivation and slight diarrhea was noted in male and female rats at 1000 mg/kg/day, occasionally during Weeks 1 and 2 and persistently during Weeks 3 and 4. For animals from this group retained for a 2-week recovery period, these clinical signs ameliorated and no further clinical signs were noted from Days 37 onwards.

For male and female rats treated at 100 mg/kg/day slight diarrhea was noted occasionally during Week 2 and persistently during Weeks 3 and 4. This was generally accompanied by pilo-erection and occasionally by increased salivation in female rats.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower bodyweight gains were recorded at Week 4 for male rats treated at 1000 mg/kg/day. This achieved statistical significance in comparison with control animals. Lower gains were also recorded for male rats treated at 100 mg/kg/day, however, this change did not achieve statistical significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slightly lower food consumption was recorded over the treatment period for male rats at 100 and 1000 mg/kg/day when compared to control animals. This can be related to the lower bodyweight gains observed at Week 4 in these animals.

During the recovery period, slightly higher food consumption was recorded for male and female rats at 1000 mg/kg/day in comparison with control animals.
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):
At Week 5 a significantly higher platelet count was recorded for male rats treated at 1000 mg/kg/day.

At Week 7 the platelet count was again higher for male rats at 1000 mg/kg/day, achieving significance in comparison with control animals. A significantly lower haemoglobin concentration was observed in male rats at 1000 mg/kg/day and a significantly lower eosinophil count was recorded for female rats at 1000 mg/kg/day. The changes in haemoglobin and eosinophils were small in magnitude and the majority of individual values were within the expected ranges for rats of this age and strain. Similar changes were not recorded at Week 4 and both findings were considered likely to have arisen by chance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At Week 5 a dosage-related increase in triglyceride levels was recorded for male rats, achieving statistical significance for male rats at 100 and 1000 mg/kg/day. Mean values at both dosages were approximately double the control value. Cholesterol levels were significantly lower for male rats at 10 and 1000 mg/kg/day. The intergroup differences, however, were not strictly dosage-dependent and were considered to be of a minor nature probably arising by chance rather than as an effect of treatment. Significantly lower glutamic-oxaloacetic transaminase (GOT) levels were recorded for males at 1000 mg/kg/day and, to a lesser extent, at 100 mg/kg/day. Although significantly lower glutamic-pyruvic transaminase (GPT) activities were observed among females at all doses, the magnitude of these changes was small and the majority of individual values were within the expected range for rats of this age and strain. The total protein and globulin levels for male rats at 1000 mg/kg/day were significantly increased. A significantly lower albumin/globulin (A/G) ratio was therefore recorded for these rats. Although the A/G ratio was also significantly lower for males at 100 mg/kg/day, there was no apparent disturbance of the separate albumin or globulin fractions. There was a tendency for lower glucose levels for male and female rats at 100 or 1000 mg/kg/day, the difference showing significance for females at 1000 mg/kg/day. These changes were, however, minor with individual values falling within the expected range for rats of this age and strain. A significantly higher calcium ion concentration was recorded for male rats at 1000 mg/kg/day. The majority of individual values were within the expected range for rats of this age and strain and this finding was considered likely to have arisen by chance.

Following the 2-week recovery period all biochemical parameters were similar for rats at 1000 mg/kg/day compared to controls.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
At Week 5 significantly higher protein concentrations were recorded for males and females at 1000 mg/kg/day.

At Week 7 mean protein concentration was higher for male rats at 1000 mg/kg/day. Although this was mainly due to 1/5 males showing a particularly high value, 2/15 other males also showed values slightly above those of control males. This change did not achieve statistical significance, however, it is possible that these differences represented a continuation of the effect seen at Week 5.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At termination, statistical analysis of liver weight data showed significantly higher values for males and females at 1000 mg/kg/day and for females at 100 mg/kg/day. In terms of absolute (unadjusted) values, however, the mean values for females at 100 mg/kg/day were similar to that of controls, whereas at 1000 mg/kg/day absolute values were also higher than controls. For male rats at 1000 mg/kg/day significantly higher adjusted kidney weights were recorded. The absolute mean kidney weights, however, were similar and the difference in adjusted values was possibly secondary to the bodyweight differences.

Following the 2-week recovery period, the adjusted liver weights were again significantly higher for male rats at 1000 mg/kg. Significantly lower adjusted brain weights were recorded for female rats at 1000 mg/kg/day. This apparent change was small in magnitude and was not considered to be related to treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Distended caeca occurred in all rats at 1000 mg/kg/day, and in 5 males and 1 female at 1000 mg/kg/day. After the 2-week recovery period, distended caeca were noted for all 5 males and 4 females at 1000 mg/kg/day. All other microscipic abnormalities were considered to be incidental and not related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal hyperplasia of the mucosal epithelium of the caeca was observed in 3 out of 5 males at 1000 mg/kg/day at the end of the 4-week treatment period. No microscopic changes were observed which could account for the relatively minor increases in liver and kidney weights. The remaining microscopic changes observed in the tissues examined were considered to be spontaneous in origin and of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Based on the statistical decrease in BW (in males) and liver and kidney weight effects at 1000 mg/kg/day, the NOAEL was 100 mg/kg/day.
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Critical effects observed:
no
Conclusions:
Male and female NOAEL = 100 mg/kg/day
Male and female LOAEL = 1000 mg/kg/day
Executive summary:

The test substance was formulated daily as a suspension in 1% methylcellulose and administered to rats by oral gavage at target doses of 1000, 100, and 10 mg/kg/day for a minimum of 28 consecutive days. Control rats received 1% methylcellulose alone. Animals were killed following the 28-day period or, for additional animals in the control and high dosage groups, following a further 2-week post-treatment recovery period. Effects of treatment observed at 100 or 1000 mg/kg/day were of a minor nature, not considered to represent serious damage to health, and were possibly associated with an established laxative action of the compound. The dosage of 10 mg/kg/day was a clear no effect level.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

The NOAEL following daily dosing for 28 days was 100 mg/kg/d; adverse effects were noted only at the top dose level of 1000 mg/kg/d.

Justification for classification or non-classification

Based on the high exposure concentration necessary to produce adverse effects, the substance does not need to be classified for repeated dose toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.