Registration Dossier

Administrative data

Description of key information

NOAEL = 1000 mg/kg bw/day (OECD 408, GLP, K, rel.1)

NOAEL = 1000 mg/kg bw/day (OECD 407, GLP, K, rel. 1)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 February 2018 to 05 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to the OECD TG No. 408 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is a suitable rodent species for toxicity testing, acceptable to regulatory authorities and for which extensive background data are available.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 to 6 weeks of age on arrival
- Weight at study initiation: males :166 to 217 g; females: 119 to 165 g
- Fasting period before study:
- Housing: Males were housed in groups of up to 3 and females were housed in groups of 5, by sex, in solid-floor cages with appropriate bedding provided. Bedding was changed as often as necessary to maintain hygiene.
- Diet: pelleted diet, ad libitum (LabDiet 5L0S EURodent Diet (manufactured by PMI Nutrition International) supplied by International Product Supplies)
- Water: ad libitum (main tap water)
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for diet and water are held centrally and retained within the Sequani archives. It was considered that none of the contaminants that were monitored was present at a level that might have prevented the study objective from being achieved.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C to 23 °C
- Humidity (%): 40 % to 70 %,
- Air changes (per hr): air-conditionned
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2018-02-22 To: 2018-06-27
Route of administration:
oral: gavage
Details on route of administration:
Based on previously performed studies.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % (w/v) (high viscosity).
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated within the known stability period, for each group separately, as a suspension in 0.5 % (w/v) carboxymethylcellulose (high viscosity).
In a fume hood, a weighed quantity of test item was added to the final preparation container with approximately 20 % of the final required volume of vehicle added and stirred vigorously to form an emulsion. After further addition of vehicle and mixing, the resultant suspension was mixed with a laboratory homogeniser and then stirred for a minimum of 20 minutes and a maximum of 168 minutes.
Formulations were divided into daily aliquots and were stored refrigerated (2 °C to 8 °C) before dosing on the day of use. They were stirred vigorously from at least 15 minutes before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on previous toxicology studies
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability: Stability of test item formulations prepared at concentrations of 1 to 100 mg/mL, spanning those used in this study (10 to 100 mg/mL), were examined in an earlier formulation validation study. Those formulations were found to be stable for 8 and 14 days when stored at room temperature (15 to 25 °C) and refrigerated (2 to 8 °C), respectively.
- Homogeneity and achieved concentrations: Two replicate sample sets were taken from the top, middle and bottom of each test item formulation prepared for use during Week 1 of dosing and were stored refrigerated (2° to 8 °C). Samples were analysed for the test item using a validated method to confirm homogeneity and achieved concentrations. As the low and intermediate dose formulations prepared for use in Week 1 did not meet the acceptance criteria for the analytical method, the second set of replicate samples from Week 1 were shipped to the Test Site and analysed; the original results were confirmed. The formulation method, sampling, transport and analytical method were reviewed and all processes were followed appropriately. As samples were transferred to the Test Site in glass containers and, at the Test Site, the whole sample was transferred and the vial rinsed into the volumetric flask it was thought that a larger shipment vial may alleviate the issues. Consequently, all subsequent samples were taken into larger vials.
Two replicate samples sets were taken in Weeks 8, 10/11, 12/13 and 13 to assess achieved concentration and confirm homogeneity. In order to assess variables in extraction and sampling during the Week 12/13 sampling, 2 replicate sample sets for each of the following were taken
as follows:
- after the homogenisation step into glass vials
- after the homogenisation step into volumetrics
- after the last stirring step into glass vials
- after the last stirring step into volumetrics
The samples taken into volumetrics were then extracted (acetone) at Sequani and the extract shipped along with the samples taken into glass vials for analysis.
In addition, samples taken from the vehicle used to dose Controls during Weeks 1, 8, 10/11 and 13 were analysed to confirm absence of test item.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 + 5 in Groups 1 and 4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on previously performed studies
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: 5 highest numbered animals of each sex from Groups 1 and 4
- Post-exposure recovery period in satellite groups: 4-weeks
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and morbidity and daily for clinical signs of toxicity or changes in behaviour or appearance

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekle

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of treatment and then weekly until necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
The amount of food consumed by each cage of animals was recorded weekly during the treatment and treatment-free periods.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in Week 13
- Dose groups that were examined: Control and high dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13 for main animals, towards the end of the treatment for satellite animals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes / No / Not specified
- How many animals: all
- Parameters checked in table [7.5.1/1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 for main animals, towards the end of the treatment for satellite animals.
- Animals fasted: Yes (isoflurane)
- How many animals: all
- Parameters checked in table [7.5.1/1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Standard arena observations: once pre-dose for all animals and once weekly thereafter, on animals designated to be killed at the end of the treatment period.
Functional observation battery: end of the treatment period
- Dose groups that were examined: all groups (excl. satellite)
- Battery of functions tested: standard arena observations and functional observation battery (sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli,
grip strength and motor activity).
- Satellites animals: grip strenght was recorded at the end of the treatment-free period.

IMMUNOLOGY: Yes
- Time schedule for examinations: after necropsy
- How many animals: 5 males
- Dose groups that were examined: high dose group
- Parameters examined.: alpha-2-microglobulin using immunochemistry
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.1/2)

HISTOPATHOLOGY: Yes (see table 7.5.1/2)
Other examinations:
None
Statistics:
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, comparing Groups 2, 3 and 4 against Group 1.
Depending on the nature of the data set that was to be analysed, appropriate tests were applied. Where parametric tests were appropriate they were preceded
by a check for homogeneity of variance using the Levene test and, where available, the Shapiro Wilks test for normality. If either of these two assumptions failed, a log transformation was applied before retesting. If the transformation failed, appropriate non-parametric tests were applied.
Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity following administration of the test item. There was a single instance of piloerection noted in one Control male.
Administration of the test item at 300 or 1000 mg/kg/day to males and females was associated with salivation following dosing. The number of animals affected generally increased with increasing dose and, at both dose levels, there were generally more females affected than males. This is a normal response to gavage dosing of an unpleasant tasting or odoriferous test item.
Other findings of wet or dry lesions, scabbing and hairloss seen across groups, including the Control group were considered to be unrelated to administration of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no early deaths following administration of the test item
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males given 1000mg/kg/day there was a 10 % reduction in mean terminal body weight and a 16 % reduction in overall body weight gain (Week 1 to Week 14), which were statistically significant (p≤0.01).
Males given 300 mg/kg/day showed a slight reduction in overall body weight gain (10 % reduction; p≤0.05) but no significant difference in terminal body weight compared with Controls.
There was no effect on body weight or body weight gain in males given 100 mg/kg/day or in females given the test item at any dose level.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects on food intake following administration of the test item to males. Group mean food intake in females given 1000 mg/kg/day was marginally but consistently higher than the Control mean throughout the dosing period. During the treatment-free period, food intake values for females previously given 1000 mg/kg/day were similar to those of the Controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular changes or abnormalities considered to be related to administration of the test item were observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In males, mean prothrombin time was shortened by 0.6 seconds at 300 mg/kg/day (p≤0.05) and by 1.4 seconds at 1000 mg/kg/day (p≤0.001) compared with the Control mean. At 1000 mg/kg/day, 7 out of 10 individual values were below the lower limit of the historical background range.
There were very small but statistically significant 2 % to 6 % reductions (p≤0.05 to p≤0.001) in mean red blood cell count, haemoglobin concentration and packed cell volume in males and females given the test item compared to Control means. However, due to their marginal nature and as all individual values were within historical background ranges, these intergroup differences were considered to be non-adverse.
There were statistically significant reductions (p≤0.05) in absolute basophil counts in females given 300 or 1000 mg/kg/day compared with the Control mean. However, as all individual values were within the historical background range, these intergroup differences were also considered to be non-adverse.
Group mean absolute lymphocyte counts in males at 300 and 1000 mg/kg/day and in females at all dose levels were statistically significantly lower (p≤0.05) than the Control means. However, most of the individual values were within the historical background range and, as these intergroup differences were not strictly related to dose, they were considered unlikely to be related to treatment with the test item.
At the end of the treatment-free period, mean prothrombin time, red cell count, haemoglobin concentration, packed cell volume, basophil and lymphocyte counts in animals previously given 1000 mg/kg/day was similar to the Control mean and there were no statistically significant intergroup differences.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males given 1000 mg/kg/day, mean plasma alkaline phosphatase (ALP) activity was statistically significantly increased (p≤0.001) compared with the Control mean, with 8 out of 10 individual ALP values above the upper limit of the historical background range. This change correlated with diffuse hepatocyte hypertrophy seen in the liver of these animals. A smaller, statistically significant increase (p≤0.05) in ALP activity was also seen in females given 1000 mg/kg/day. However, in this case, most of the individual findings were within the historical background range.
Mean plasma albumin concentration was statistically significantly increased (p≤0.001) and mean plasma globulin concentration was decreased (p≤0.001) in males at 1000mg/kg/day, resulting in a statistically significant increase (p≤0.001) in mean albumin/globulin ratio (A/G ratio) compared with the Control mean. All of the individual values for albumin and A/G ratio were above the upper limit of the historical background range while 9 out of 10 individual globulin values were below the lower limit of the background range. Similar smaller changes were seen in these parameters (p≤0.05 to p≤0.01) in males given 300 mg/kg/day and, for A/G ratio, in females given 1000 mg/kg/day (p≤0.05) in comparison with Controls. In these instances, most of the individual values were within the historical background ranges.
Other statistically significant non-adverse intergroup differences included increases in plasma urea concentration in males and females given 1000 mg/kg/day, decreases in plasma cholesterol and triglyceride concentrations in males given 300 or 1000 mg/kg/day and decreases in plasma calcium concentrations at all test item dose levels. In all these instances, most or all of the individual values were within the historical background ranges.
At the end of the treatment-free period, mean plasma calcium concentration in males previously given 1000 mg/kg/day remained slightly lower (p≤0.05) than the Control mean. The means for all other parameters that were affected by treatment were similar to the Control means. Mean plasma potassium concentrations in males and females previously given 1000 mg/kg/day were significantly different from the Control means. However, in males they were slightly increased
while in females they were slightly decreased. As the results are contradictory and as this parameter was unaffected at the end of the treatment period, this finding is considered to be spurious and unrelated to previous treatment with the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Standard arena observations: Other than excessive salivation seen in one high dose male and one high dose female, there were no findings in the standard arena or functional observations battery of tests that were
considered to be test item related. Other findings occasionally seen in Controls as well as the test item treated animals were either apparent before the start of dosing or affected only one or two animals in a group and were therefore considered to be unrelated to treatment with the test item.
- Grip strength: There were no effects on grip strength that were clearly related to the test item. Towards the end of the treatment period, mean hindlimb grip strength in males was statistically significantly lower (p≤0.01 to p≤0.001) than the Control mean at all dose levels. However, mean forelimb grip strength was only lower than the Control mean (p≤0.05) in the males at 1000 mg/kg/day. There was a great deal of inter-animal variation within and between groups and as the lower forelimb grip strength in males was confined to the high dose group only and in the absence of evidence of neurological pathology changes, it was considered that these intergroup differences in males were unlikely to be related to administration of the test item. At the end of the treatment-free period, there were no statistically significant differences in mean forelimb or hindlimb values between males or females previously given 1000 mg/kg/day and the Controls.
- Motor activity: Administration of the test item had no effect on motor activity. At the end of the treatment period, there were statistically significantly lower (p≤0.05) movement counts and distances travelled with a corresponding higher time at rest (p≤0.05) for males given 1000 mg/kg/day during one time period only (Period 5) compared with Controls over the same period. As these differences were confined to just one period, they were considered to be unrelated to treatment with the test item. In females given 300mg/kg/day, there were statistically significantly lower (p≤0.05) movement counts and distances travelled with a corresponding higher time at rest (p≤0.01) during one period only (Period 4) compared with Controls over the same period. As these differences were confined to just one period and were not evident at 1000 mg/kg/day, they also were considered to be unrelated to treatment with the test item.
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
Sections of kidneys from five high dose males (including those showing the most hyaline droplets) were stained for α2u-globulin using immunohistochemistry. The sections were treated with a specific mouse anti-rat α2u-globulin antibody (R&D Systems catalogue number MAB586-100). Sections of male rat kidneys, known to be positive for α2u-globulin, were treated similarly and used as Positive Controls.
Microscopic examination revealed that the test item-related change seen in the routine H&E stained sections was not shown by the immunohistochemical staining, indicating that the increased number of hyaline droplets was not positive for α2u-globulin. Thus, the mechanism of the increased incidence/severity of hyaline droplets seen within the cortical tubules of male kidneys in this study remained unestablished.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant 29 % to 50 % increases (p≤0.001) in mean absolute and body weight related liver weights were seen in males and females given 1000 mg/kg/day, consistent with the diffuse hepatocyte hypertrophy seen in the liver of these rats. In addition, smaller 13 % increases (p≤0.01) in mean body weight related liver weight were also seen in males given 300 mg/kg/day. All of the individual relative liver weights from males and females given 1000 mg/kg/day were above the upper limit of the historical background range whereas all of the individual values for animals given 300 mg/kg/day were within the background range.
At 1000 mg/kg/day, there were statistically significant 14 % to 36 % increases (p≤0.05 to p≤0.001) in mean absolute or body weight related kidney weight in males and females compared with the Control means. In addition, in males given 300 mg/kg/day, mean relative kidney weight was also increased (p≤0.001) compared with the Control mean. In the high dose males, 5 out of 10 individual relative kidney weights were above the upper limit of the historical background range. All or most individual relative kidney weights in animals given lower doses were within the background range.
At 1000 mg/kg/day, mean absolute (females only) and body weight related (males and females) thyroid weight was 23 % to 50 % higher (p≤0.01 to p≤0.001) than the Control means, although all individual values were within the historical background range. Generally smaller increases (p≤0.05) were also seen in both sexes at 300 mg/kg/day and in females only at 100 mg/kg/day. These weight differences were not associated with any histopathology changes in the thyroid gland and were therefore considered to be non-adverse.
Mean ovary weights in females given 300 or 1000 mg/kg/day were 18 % to 21 % higher (p≤0.05) than the Control mean. As there was no histopathological evidence to account for these weight changes, they were considered to be non-adverse.
Minor (7 %; p≤0.05) decreases in mean absolute epididymides weight seen at 300 and 1000 mg/kg/day were considered not to be related to treatment with the test item and small statistically significant increases in mean body weight related testes weight compared with Controls were due to intergroup differences in mean terminal body weights.
At the end of the treatment-free period, mean liver and ovary weights in males and females and kidney weights in females previously given 1000 mg/kg/day were similar to Control means, with no statistically significant intergroup differences. However, in males, mean body weight related kidney weight and absolute and body weight related thyroid weights remained statistically significantly higher (p≤0.05) than the Control means. The difference in relative kidney weight and the statistical probability were reduced in the treatment-free period group compared to the end of treatment group, which may indicate some degree of reversibility.
Mean body weight related spleen weight in females previously given 1000 mg/kg/day was higher (p≤0.05) than the Control mean. However, as spleen weight was unaffected at the end of the treatment period, this result at the end of the treatment-free period was considered to be spurious and unconnected to treatment with the test item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, there were test item-related macroscopic findings in the liver and kidneys in males from all test item treated groups.
There was a low incidence of kidneys showing abnormal discolouration, mainly described as mottled, in all groups of males given the test item (3 out of the 10 animals from all groups). This finding correlated with the microscopic findings (tubular, granular casts) and therefore was considered to be test item-related.
One male given test item at 1000 mg/kg/day had an abnormally large liver and this correlated with test item-related microscopic findings (diffuse hepatocyte hypertrophy).
The spectrum of other macroscopic findings was consistent with those normally encountered in rats of this age and strain kept under laboratory conditions.
At the end of the treatment-free period, there were no test item-related macroscopic findings in the liver and kidneys, which suggests reversibility of the effects in these organs.
Neuropathological findings:
not examined
Description (incidence and severity):
Test item-related microscopic findings were seen in the livers of all males and some females given test item at 1000 mg/kg/day and in the kidneys of males given test item at 100, 300 or 1000 mg/kg/day.
- LIVER (Table 7.5.1/3): Diffuse hepatocyte hypertrophy (slight) was found in all males and 4 females given test item at 1000 mg/kg/day. This finding correlated with the increase in liver weight in rats of the high dose group (1000 mg/kg/day), when compared with Controls.
- KIDNEYS (Table 7.5.1/4): Hyaline droplets were seen at a low level in Control males, but there was an increased incidence/severity in males given test item at all dose levels, which was considered to be test item-related. Hyaline droplets were not seen in the tubular epithelium in females. In addition, an increased incidence/severity of basophilic cortical tubules and tubular, granular casts at the cortico-medullary junction were found only in male rats given test item.
- OTHER FINDINGS: The spectrum of other microscopic findings was consistent with those normally encountered in rats of this age and strain kept under laboratory conditions. In particular, there was no histopathological evidence to account for the increased weights of thyroid glands (in males given 300 or 1000 mg/kg/day and females from all test item treated groups) or for the increased weights of ovaries in females given 300 mg/kg/day and 1000 mg/kg/day at the end of the treatment period.

TREATMENT FREE PERIOD: At the end of the treatment-free period, there were still histopathological findings seen in the kidneys of all males, whereas the change in the liver (diffuse hepatocyte hypertrophy) showed full reversibility within the treatment-free period.
- KIDNEYS (Table 7.1.5/5): At the end of the treatment-free period, there was still an increased incidence/severity of basophilia in the tubular cortex and tubular granular casts at the cortico-medullary junction in animals previously given 1000 mg/kg/day when compared with the Controls. The incidence levels of these findings were similar to those seen at the end of the treatment period and there was no evidence of recovery. The incidence/severity of hyaline droplets was similar in Control and treated animals and therefore recovery of this finding was achieved within the treatment-free period.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed
Key result
Critical effects observed:
no

Table 7.5.1/3: Microscopic pathology findings: Liver

 

Males

Females

Group

1

2

3

4

1

2

3

4

Dose level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Number of rats examined

10

10

10

10

10

10

10

10

Liver

 

 

 

 

 

 

 

 

 

Hypertrophy, hepatocyte, diffuse

Slight

0

0

0

10

0

0

0

4

Total

0

0

0

10

0

0

0

4

Table 7.5.1/4: Microscopic pathology findings: Kidneys

 

Males

Group

1

2

3

4

Dose level (mg/kg/day)

0

100

300

1000

Number of rats examined

10

10

10

10

Kidneys

 

 

 

 

 

Hyaline droplets, tubular

Minimal

7

4

1

0

Slight

0

5

7

2

Moderate

0

0

2

8

Total

7

9

10

10

 

 

 

 

 

 

Basophilia, tubular, cortex, focal

Minimal

4

3

4

5

Slight

0

2

1

2

Moderate

0

0

5

3

Total

4

5

10

10

 

 

 

 

 

 

Casts, tubular, granular, cortico-medullary junction

Minimal

0

3

1

5

Slight

0

1

1

1

Moderate

0

1

5

3

Marked

0

1

1

1

Total

0

6

8

10

Table 7.5.1/5: Microscopic pathology findings: Kidneys (recovery group):

 

Males

Group

1

2

3

4

Dose level (mg/kg/day)

0

100

300

1000

Number of rats examined

5

-

-

5

Kidneys

 

 

 

 

 

Hyaline droplets, tubular

Minimal

4

-

-

4

Total

4

-

-

4

 

 

 

 

 

 

Basophilia, tubular, cortex, focal

Minimal

1

-

-

2

Slight

0

-

-

1

Marked

0

-

-

1

Total

1

-

-

4

 

 

 

 

 

 

Casts, tubular, granular, cortico-medullary junction

Minimal

0

-

-

1

Slight

0

-

-

1

Moderate

0

-

-

1

Total

0

-

-

3

Conclusions:
Oral gavage administration of the test material to the Crl:WI(Han) strain of rat at dose levels of 0, 100, 300 or 1000 mg/kg bw/day was generally well tolerated, with no deaths or clinical signs of toxicity. The main effects of treatment were a non-adverse adaptive hepatocyte hypertrophy associated with an increase in liver weight and male rat specific α2u-globulin nephropathy, which were considered non-adverse. Administration of 1000 mg/kg bw/day to males resulted in a 10 % reduction in terminal body weight and fully reversible changes in clinical pathology parameters which were considered to be secondary to the liver hypertrophy and non-adverse. The No Observed Adverse Effect Level was therefore considered to be 1000 mg/kg bw/day.
Executive summary:

Study Objective.  To assess the potential toxicity of the test material when administered by gavage to the rat daily for at least 90 days and to assess the reversibility of any observed effects over a 4-week treatment-free period.

Methods.  Fifty male and 50 female rats of the Crl:WI(Han) strain were allocated to the study.  Groups of 10 males and 10 females were dosed with 0 (vehicle), 100, 300 or 1000 mg/kg bw/day the test material, once daily, by gavage at a dose volume of 10 mL/kg body weight for 90 days, until the day before necropsy.  A further 5 males and 5 females were added to each of the Control and high dose groups and were also dosed for at least 90 days before being retained undosed for 4-weeks to assess the reversibility of any observed effects.  

All animals were observed daily from the start of treatment and body weights and food intake were recorded at weekly intervals until the day of necropsy. Any potential effects of the test material on central and peripheral nervous system function were determined from the battery of standard arena and functional observation tests conducted on all animals before the start of treatment and once weekly thereafter on all animals to be subject to necropsy at the end of the treatment period. On one occasion towards the end of the treatment period, all animals designated to be subject to necropsy at the end of the treatment period were observed for sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity. In addition, animals allocated to the treatment-free period were observed for grip strength towards the end of the treatment-free period. The eyes of all animals were examined before the start of treatment, with Control and high dose animals also being examined in Week 13.  

Blood samples were taken during Week 13 and towards the end of the treatment-free period for clinical pathology.  Animals were subjected to a gross necropsy, specified organs were weighed and a full list of tissues was examined microscopically from the Control and high dose animals at the end of the treatment period.  In addition, the livers from all males and females and the kidneys from all males in the low and intermediate dose groups and those animals subjected to necropsy at the end of the treatment-free period were also examined.

Results.  Oral administration of the test material for 90 days was generally well tolerated with no deaths and no clinical signs of toxicity. There were no adverse effects on peripheral nervous system function or on sensorimotor responses to stimuli, grip strength, motor activity or food intake or ocular changes in either sex or body weight effects in females. There was a 10 % reduction in terminal body weight in males given 1000 mg/kg bw/day. Findings in the kidneys of males at all the test material dose levels were indicative of α2u-globulin nephropathy (granular, tubular casts at the cortico-medullary junction; increased incidence/severity of tubular basophilia and increased incidence/severity of hyaline droplets). However, as this is known to be specific to male rats and of no relevance to humans, it was considered not to be adverse. Administration of 1000 mg/kg bw/day resulted in a reversible, non-adverse adaptive diffuse hepatocyte hypertrophy in all males and some females, associated with an increase in liver weight in both sexes. This hypertrophy was associated with a number of fully reversible secondary non-adverse effects including a shortening of prothrombin time, an increased plasma albumin/globulin ratio due to an increased albumin and decreased globulin concentrations and an increase in plasma alkaline phosphatase activity.

Conclusions.  Oral gavage administration of the test material to the Crl:WI(Han) strain of rat at dose levels of 0, 100, 300 or 1000 mg/kg bw/day was generally well tolerated, with no deaths or clinical signs of toxicity. The main effects of treatment were a non-adverse adaptive hepatocyte hypertrophy associated with an increase in liver weight and male rat specific α2u-globulin nephropathy, which were considered non-adverse. Administration of 1000 mg/kg bw/day to males resulted in a 10 % reduction in terminal body weight and fully reversible changes in clinical pathology parameters which were considered to be secondary to the liver hypertrophy and non-adverse. The No Observed Adverse Effect Level was therefore considered to be 1000 mg/kg bw/day.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in rats.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1995-03-24 to 1995-05-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to EU Method B.7 and in compliance with GLP.
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1992
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Storage conditions: 4 °C in the dark
Species:
rat
Strain:
Crj: CD(SD)
Remarks:
Crl:CD(SD)BR (VAF plus)
Details on species / strain selection:
The animal species was chosen by the Sponsor
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles river (UK) Limited, Margate, England
- Age at study initiation: about 28 days old on arrival
- Weight at study initiation: males 159-197 g, females 132-170 g
- Fasting period before study: none
- Housing: in groups of 5, by sex, in grid-bottomed stainless steel cages
- Diet (e.g. ad libitum): pelleted diet (SQC Rat and Mouse Maintenance Diet No. 1, Expanded, Special Diets Services Limited, Witham, England) ad libitum, unless period of deprivation associated with laboratory investigations
- Water (e.g. ad libitum): ad libitum, unless period of deprivation associated with laboratory investigations
- Acclimation period: 7-14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-26 °C. The temperature fell below the protocol specified lower limit of 19 °C on six occasions, falling to 17 °C. A temperature of 26 °C was recorded on only one occasion.
- Humidity (%): 32-59 %
- Air changes (per hr): air conditioned
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 1995-03-24 to 1995-05-04
Route of administration:
oral: gavage
Details on route of administration:
The route of administation was considered to ensure an absorption at least equal to that of the percutaneous application which is the route of use in humans.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 %
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was formulated daily for dosing as suspensions in the vehicle (0.5 % w/v CMC). Separate formulations were prepared for each dose level. The weighted quantity of test article was suspended in the appropriate volume of vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test material is not soluble in water
- Concentration in vehicle: 0.5 % w/v
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation were analysed for test material on one day in weeks 1 and 4.
A gas chromatographic method of analysis (Toxicol reference, FCH002) was developed and validated. The detector response of ST 06 C 93 was found to be linear over the injection concentration range of 53.0 µg/mL to 285.5 µg/mL. The accuracy/precision of analysis at concentration of 5 and 100 mg/L in 0.5 % CMC was determined to be 106% / 0.52% and 101% / 0.63% respectively.
The intra accuracy and precision (homogeneity) of formulation at concentration of 25 and 100 mg/mL in 0.5% CMC was determined to be 94% / 6.11% and 98% / 4.70% respectively. The inter accuracy / precision of formulation at concentration of 25 and 100 mg/mL was determined to be 91% / 3.58% and 98% / 4.13% respectively.
The test article formed a homogenous suspension in the specified vehicle (0.5 %, w/v aqueous CMC) which was chemically stable for at least 24 hours at concentrations of 25 and 100 mg/mL.
The concentration of ST 06 C 93 in study formulation analysed on week 1 and 4 were within 10 % of their nominal concentration. Test article formulations were therefore considered to be suitable for the purpose of the study.

Duration of treatment / exposure:
4 weeks
Frequency of treatment:
once daily
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in light of the study "7-d oral (gavage) dose range-finding study in the rat". 24 rats of the Crl:CD(SD)BR strain were divided into 4 groups consisting of 3 males and 3 females. Three groups received the test material orally by gavage once daily, for 7 days at dose levels of 500, 750 or 1000mg/kg bw/day. The fourth group received the vehicle 0.5% (w/v) aqueous CMC and acted as control. Administration of ST 06 C 93 orally by gavage was associated with increased absolute and bodyweight related liver weights in males. In the absence of any findings at necropsy, it was concluded that 1000 mg/kg bw/d would however be a suitable high dose level for a long term study. Due to the slightly higher liver weight in male animals at 500 mg/kg bw/d, the low dose level selected for the 28-d study was 250 mg/kg bw/d.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: satellite groups were not included
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study and then weekly thereafter.

FOOD CONSUMPTION:
The food consumed by each cage of animals was recorded weekly throughout the treatment and group mean weekly intakes were calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes - Both eyes of all animals were examined before treatment started.
- Time schedule for examinations: in week 4
- Dose groups that were examined: the eyes of all control and high dose animals were examined (Group 1 and 4).
- Examinations: using an indirect ophtalmoscope and then, if necessary, a direct ophtalmoscope after previous instillation of a mydriatic agent (Minims - 2% w/v Homatropine hydrobromide, Smith and Nephew Pharmaceuticals Limited, Romfort, England).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once in week 4
- Anaesthetic used for blood collection: No data, as the blood samples were taken by tail venepuncture.
- Animals fasted: Yes, overnight for haematology; for coagulation tests animal were not fasted.
- How many animals: all
- Parameters checked in Table 7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 4 for clinical chemistry and in week 5 for coagulation; and at the end of recovery period for groups 1 and 4
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked in table 7.5.1/1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: end of week 3
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 7.5.1/1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.1/2)
HISTOPATHOLOGY: Yes (see table 7.5.1/2)
Other examinations:
None
Statistics:
ANOVA was performed on all parameters, using treatment group as the factor in the analysis. Residuals from this preliminary analysis were examined for heterogeneity of variance using Levenes tests. If Levenes test was significant at the 1% level then the particular variable concerned was subjected to a non-parametric analysis. Otherwise, Williams test was performed to compare the high dose with control at the two-sided 5% level. If this test was statistically significant then comparisons of the subsequent doses against control were performed at the one-sided 5% level until a non-significant difference is found, at which point the testing was stopped. The highest dose at which the difference from control was non-significant was deemed to be the no effect level.
If Levenes test indicated that there were differences in the treatment group variances, then a Kruskal-Wallis ANOVA was performed to assess overall differences amongst the treatment groups, followed by Shirley’s non-parametric version of William’s test, which was based on mean ranks rather than the arithmetic means. Note that the Shirley’s test was the modified version described in Williams which should enhance the power of the tests of the lower doses against control, and was performed in the same hierarchical manner as Williams test above.
Variables that were measured pre-study (for example body weight prior to the first administration of treatment) were analysed by first performing ANOVA, and then assessing the heterogeneity of the treatment group variances using Levenes test and performing a non-parametric ANOVA analysis, if appropriate. This provided an overall test for the comparability of the treatment groups, and was followed by all possible pairwise tests using the protected Student t-test (or its non-parametric equivalent).
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment related clinical signs noted. The only clinical sign noted was dorsal cervical hairloss in males dosed with 250 mg/kg bw/d on days 7-29.
Mortality:
no mortality observed
Description (incidence):
There were no deaths during the treatment period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Unaffected by treatment
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Unaffected by treatment
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings or abnormalities
Haematological findings:
no effects observed
Description (incidence and severity):
See Table 7.5.1/5
Neutrophil levels were higher and lymphocyte counts were lower in males treated with 500 and 1000 mg/kg bw/d compared to the controls. These values were at the upper or lower limit of the normal ranges respectively. The toxicological significance of these findings cannot be discounted.
A small number of other statistically changes were noted, however all changes were within the normal ranges found in the laboratory.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Mean glucose levels were reduced in all treated male groups and high dose group females. As these values were within the normal ranges found in the laboratory and / or did not occur in a dose related manner they were considered not to be of toxicological significance.
All other statistically changes were within the normal ranges found in the laboratory and were considered to be unrelated to treatment
Urinalysis findings:
no effects observed
Description (incidence and severity):
One medium-dose and two high-dose males showed moderate levels of ketones.
There were no other effects on urine composition or cellularity.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
See Table 7.5.1/3
Absolute and bodyweight related liver weights were statistically increased in males dosed at 500 mg/kg bw/d and both sexes at 1000 mg/kg bw/d compared to control values. The actual values for mean liver weight and mean bodyweight-related liver weight in these groups were outwith the normal ranges found in this laboratory.
A statistically significant increase in absolute and bodyweight-related thyroid weights was apparent in males dosed at 1000 mg/kg bw/d. All treated females showed an increase in bodyweight related thyroid weights. Group mean values were within the normal ranges found in the laboratory.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
See Table 7.5.1/4
Microscopic examination revealed an increased incidence of minimal kidney tubular basophilia in animals dosed at 1000 mg/kg bw/d compared to controls.
The remaining small numbers of histopathological findings recorded were within the normal range of background alterations which may be seen in rats of this age and strain.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Critical effects observed:
no

Table 7.5.1/3: Liver and thyroid weights:

Dose level (mg/kg bw/d)

Liver

Thyroid

Weight (g)

% of bodyweight

Weight (g)

% of bodyweight

Males

0

Mean

SD

17.85

1.205

4.86

0.212

16

1.4

4.38

0.611

250

Mean

SD

18.70

1.197

5.18

0.542

15

3.0

4.25

0.745

500

Mean

SD

20.49

2.443

5.82**

0.311

15

0.4

4.34

0.290

1000

Mean

SD

25.24***

4.788

7.04***

0.546

19*

0.5

5.56*

0.832

Females

0

Mean

SD

10.04

0.298

4.29

0.218

11

1.6

4.72

0.861

250

Mean

SD

9.18

0.525

3.93

0.233

12

1.3

5.21

0.546

500

Mean

SD

10.80

1.051

4.52

0.217

13

2.8

5.59

1.445

1000

Mean

SD

11.58

1.132

5.29***

0.333

13

1.6

5.74

0.631

*= significantly different from control value (p<0.05), Williams test

** = significantly different from control value (p<0.01), Williams test

*** = significantly different from control value (p<0.001), Williams test

Table 7.5.1/4 : Histopathological findings

Histopathological Finding

Dose Level (mg/kg bw/d)

Males (5/dose)

Females (5/dose)

0

250

500

1000

0

250

500

1000

Kidney

Tubular basophilia

-         Minimal

1

1

1

3

0

0

0

2

Tubular dilatation

-         Minimal

0

0

1

1

0

0

0

0

Chronic inflammation

-         Minimal

0

0

1

0

0

0

0

0

Mononuclear cell infiltration

-         Minimal

1

0

0

0

0

2

0

1

Pelvic dilatation

-         Slight

0

1

0

0

0

0

0

0

Congestion

-         Slight

0

1

0

0

0

0

0

0

Tubular mineralisation

 0

 0

 0

0

0

1

3

Pyelitis

-         Moderate

0

0

0

0

0

0

1

0

 

Liver

Congestion

-         Slight

0

-

1

0

0

-

-

0

-         Moderate

0

-

0

3

0

-

-

0

 

Table 7.5.1/5: Haematology findings

 

Dose level (mg/kg bw/d)

Leukocyte differential count (% WBC)

Neutrophils

Lymphocytes

Mean

SD

Mean

SD

Males

0

12

3.4

84

3.7

250

13

4.9

84

6.1

500

23**

10.0

74*

9.9

1000

26**

2.9

71**

2.2

Females

0

19

4.2

76

3.6

250

12

6.7

83

6.5

500

17

6.5

79

5.8

1000

25

8.4

22

6.9

*= significantly different from control value (p<0.05), Williams test

** = significantly different from control value (p<0.01), Williams test


 

Conclusions:
Under the test conditions, the NOAEL for systemic effects was determined to be 1000 mg/kg bw/day based on the absence of effects with toxicological concern.
Executive summary:

In a subacute oral toxicity study performed according to EU test method B.7 and in compliance with GLP, ST 06 C 93 diluted in carboxymethylcellulose was administered to 5 Crl: CD (SD) BR (VAF plus) rats/sex/dose by gavage at dose levels of 0, 250, 500 and 1000 mg/kg bw/day for 28 days. Control rats were given the vehicle alone.

The dose levels were selected based on the result on a seven-day preliminary study. Administration of the test material was associated with increased absolute and bodyweight related liver weights in males at all dose levels (500, 750 and 1000 mg/kg bw/day). In the absence of any findings at necropsy, it was concluded that 1000 mg/kg bw/day would be a suitable high dose level for a longer study. Due to the slightly higher liver weight in male animals at 500 mg/kg bw/day, the low dose level selected for the 28-d study was 250 mg/kg bw/day.

 

Animals were observed daily and body-weight and food consumption was recorded weekly. Ophtalmoscopy was performed on both eyes of all animals before the start of treatment and on control and high dose groups during week 4. Blood samples were obtained for all animals during week 4 and urine samples were collected from all animals at the end of week 3. At termination all animals were subjected to a detailed necropsy during which the weight of identified organs were recorded and a wide range of tissues preserved. A microscopic examination of specified tissues was performed from all control and high dose animals. All gross lesions and kidneys were examined from all animals.

 

No mortality was observed in any group. There were no treatment related clinical signs noted. Bodyweight, bodyweight gains and food consumption were not adversely affected by treatment. There were no treatment-related ocular findings. At necropsy, all organs and tissues appeared normal during the macroscopic observations. Blood chemistry did not measure any changes which were considered to be of toxicological significance. Urinary parameters were unaffected by administration of the test material.

 

Haematological evaluation revealed a higher mean neutrophil levels and lower mean lymphocyte counts in males treated with ST 06 C 93 at 500 and 1000 mg/kg bw/day compared to the controls. Satellite groups were not included in the study design; therefore the reversibility of haematological effects cannot be evaluated. However, these values were in the normal ranges found in the laboratory, although situated at the upper or lower limit. Moreover, both parameters are affected by animal maintenance (stress) and diurnal variations and could not be regarded as indicative of an adverse effect without some other corroborative changes.

 

A statistically significant increase in absolute and bodyweight-related thyroid weights was apparent in males dosed at 1000 mg/kg bw/day. All treated females showed an increase in bodyweight related thyroid weights. However group mean values were within the historical ranges of the laboratory and these changes were not accompanied by histopathological findings. Therefore this effect was not considered to be of toxicological significance.

Absolute and relative liver weights were statistically increased in males dosed at 500 mg/kg bw/day and both sexes at 1000 mg/kg bw/day compared to control values. The actual values for mean liver weight and mean relative liver weight in these groups were outwith the normal ranges of the laboratory historical data. Slight to moderate liver congestion was observed in males dosed at 500 and 1000 mg/kg bw/day (1/5 and 3/5 respectively). These types of effects are well known and are considered to represent a demand for increased liver function indicative of a physiological adaptive response.

Microscopic examination revealed an increased incidence of minimal kidney tubular basophilia in animals dosed at 1000 mg/kg bw/day (3/5 males, 2/5 females) compared to controls (1/5 males, 0/5 females) and an increased incidence of tubular mineralisation in females dosed at 1000 mg/kg bw/day (3/5) compared to control (0/5). However, renal tubular basophilia is a relatively frequent spontaneous finding in young rats. Its incidence is higher in males than in females. It is believed that these tubular changes are early stages in chronic progressive nephrosis because they are frequently seen in strains where a high incidence of chronic progressive nephrosis occurs in older animals. The Sprague-Dawley strain is considered one of the most susceptible strains to chronic progressive nephrosis. Tubular mineralization is also a current finding in rats, specifically in female, and is thought to be related to oestrogen levels.

 

Under the test conditions, the NOAEL for systemic effects was determined to be 1000 mg/kg bw/day based on the absence of effects with toxicological concern.

This study is considered as acceptable and satisfies the requirement for sub-acute oral toxicity endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is GLP-compliant and of high quality (Klimisch score = 1)

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

Subchronic study:

A key study was identified (Sequani, 2019, Rel.1). In this subchronic oral toxicity study performed according to the OECD TG No. 408 and in compliance with GLP, the test material diluted in carboxymethylcellulose was administered to Crl:WI(Han) strain rats by gavage at dose levels of 0, 100, 300 or 1000 mg/kg bw/day for 90 days.

Oral administration of the test material for 90 days was generally well tolerated with no deaths and no clinical signs of toxicity. There were no adverse effects on peripheral nervous system function or on sensorimotor responses to stimuli, grip strength, motor activity or food intake or ocular changes in either sex or body weight effects in females. There was a 10 % reduction in terminal body weight in males given 1000 mg/kg bw/day. Findings in the kidneys of males at all the test material dose levels were indicative of α2u-globulin nephropathy (granular, tubular casts at the cortico-medullary junction; increased incidence/severity of tubular basophilia and increased incidence/severity of hyaline droplets). However, as this is known to be specific to male rats and of no relevance to humans, it was considered not to be adverse. Administration of 1000 mg/kg bw/day resulted in a reversible, non-adverse adaptive diffuse hepatocyte hypertrophy in all males and some females, associated with an increase in liver weight in both sexes. This hypertrophy was associated with a number of fully reversible secondary non-adverse effects including a shortening of prothrombin time, an increased plasma albumin/globulin ratio due to an increased albumin and decreased globulin concentrations and an increase in plasma alkaline phosphatase activity.

Oral gavage administration of the test material to the Crl:WI(Han) strain of rat at dose levels of 0, 100, 300 or 1000 mg/kg bw/day was generally well tolerated, with no deaths or clinical signs of toxicity. The main effects of treatment were a non-adverse adaptive hepatocyte hypertrophy associated with an increase in liver weight and male rat specific α2u-globulin nephropathy, which were considered non-adverse. Administration of 1000 mg/kg bw/day to males resulted in a 10 % reduction in terminal body weight and fully reversible changes in clinical pathology parameters which were considered to be secondary to the liver hypertrophy and non-adverse. The No Observed Adverse Effect Level was therefore considered to be 1000 mg/kg bw/day.

Subacute study:

A key study was identified (Toxicol, 1996, Rel.1). In this subacute oral toxicity study performed according to EU test method B.7 and in compliance with GLP, the test material diluted in carboxymethylcellulose was administered to Crl: CD (SD) BR (VAF plus) strain rats by gavage at dose levels of 0, 250, 500 and 1000 mg/kg bw/day for 28 days.

No mortality was observed in any group. There were no treatment related clinical signs noted. Bodyweight, bodyweight gains and food consumption were not adversely affected by treatment. There were no treatment-related ocular findings. At necropsy, all organs and tissues appeared normal during the macroscopic observations. Blood chemistry did not measure any changes which were considered to be of toxicological significance. Urinary parameters were unaffected by administration of the test material.

Haematological evaluation revealed a higher mean neutrophil levels and lower mean lymphocyte counts in males treated with ST 06 C 93 at 500 and 1000 mg/kg bw/day compared to the controls. Satellite groups were not included in the study design; therefore the reversibility of haematological effects cannot be evaluated. However, these values were in the normal ranges found in the laboratory, although situated at the upper or lower limit. Moreover, both parameters are affected by animal maintenance (stress) and diurnal variations and could not be regarded as indicative of an adverse effect without some other corroborative changes.

A statistically significant increase in absolute and bodyweight-related thyroid weights was apparent in males dosed at 1000 mg/kg bw/day. All treated females showed an increase in bodyweight related thyroid weights. However group mean values were within the historical ranges of the laboratory and these changes were not accompanied by histopathological findings. Therefore this effect was not considered to be of toxicological significance.

Absolute and relative liver weights were statistically increased in males dosed at 500 mg/kg bw/day and both sexes at 1000 mg/kg bw/day compared to control values. The actual values for mean liver weight and mean relative liver weight in these groups were outwith the normal ranges of the laboratory historical data. Slight to moderate liver congestion was observed in males dosed at 500 and 1000 mg/kg bw/day (1/5 and 3/5 respectively). These types of effects are well known and are considered to represent a demand for increased liver function indicative of a physiological adaptive response.

Microscopic examination revealed an increased incidence of minimal kidney tubular basophilia in animals dosed at 1000 mg/kg bw/day (3/5 males, 2/5 females) compared to controls (1/5 males, 0/5 females) and an increased incidence of tubular mineralisation in females dosed at 1000 mg/kg bw/day (3/5) compared to control (0/5). However, renal tubular basophilia is a relatively frequent spontaneous finding in young rats. Its incidence is higher in males than in females. It is believed that these tubular changes are early stages in chronic progressive nephrosis because they are frequently seen in strains where a high incidence of chronic progressive nephrosis occurs in older animals. The Sprague-Dawley strain is considered one of the most susceptible strains to chronic progressive nephrosis. Tubular mineralization is also a current finding in rats, specifically in female, and is thought to be related to oestrogen levels.

Under the test conditions, the NOAEL for systemic effects was determined to be 1000 mg/kg bw/day based on the absence of effects with toxicological concern.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Based on the available information, no self-classification is proposed according to the CLP and the GHS.