Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18-06-2018 to 24-01-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD TG 407 without any deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
- Substance type: Colourless liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light container flushed with nitrogen

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
The Spague Dawley rat was chosen as the animal model for this study as it is an accepted rodent species and strain for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9-11 weeks old at initiation of dosing
- Weight at study initiation: 230-450 g
- Fasting period before study:
- Housing: up to 5 animals (of the same sex and same dosing group together) in polycarbonate cages (Makrolon type 2000P, height 21.5 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet (e.g. ad libitum): ad libitum (except during designated procedures)
- Water (e.g. ad libitum): ad libitum (except during motor activity assessment)
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
- Food: Prepared diets were provided ad libitum in stainless steel containers, covered by a stainless steel grid to prevent spillage, except during designated procedures. Diets remained in the food hopper for a maximum of 5 days, and on the day of weighing the remaining food in the food hopper was replaced with new room temperature-acclimated diet (at least 1 hour prior to opening the diet bag) retained from the freezer. Food hoppers were shaken on a daily basis to divide any sawdust equally over the diet in order to facilitate food consumption. During motor activity measurements, animals did not have access to food for a maximum of 2 hours
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water was freely available to each animal via water bottles.
During motor activity measurements, animals had no access to water for a maximum of 2 hours.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21°C (target 18 to 24°C)
- Humidity (%): 46 to 70 % (target 40 to 70 %)
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2018-06-18 To: 2018-10-30

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The oral route of exposure via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): 5 days (maximum)
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Storage temperature of food: in the freezer (≤-15°C) until use (if not used on the day of preparation for a maximum of three weeks). Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 5 days (stability is confirmed under Test Facility Study No. 20153658 (analytical Method Development and Validation Study)) for supplementing food during the respective food consumption measurement interval.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet preparation samples were collected for analysis at 3 occasions (Week 1-6-13). Concentration was analyzed in all groups. Homogeneity was analyzed in Group 1 and 4.
- Analytical method: validated procedure (GC-FID)
- Concentration analysis: Duplicate sets of middle samples (approximately 5 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
- Homogeneity analysis: Duplicate sets of top, middle and bottom samples (approximately 5 g) for each sampling time point were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ±10%. After acceptance of the analytical results, backup samples were discarded.
- Stability analysis: Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
For a minimun of 90 days
Frequency of treatment:
Ad lilbitum
Doses / concentrationsopen allclose all
Dose / conc.:
500 ppm
Remarks:
Main group.
29 mg/kg bw/day (24-33) in males; 37 mg/kg bw/ day (28-43) in females
Dose / conc.:
5 000 ppm
Remarks:
Main group.
287 mg/kg bw/day (241-353) in males; 351 mg/kg bw/ day (297-421) in females
Dose / conc.:
11 000 ppm
Remarks:
Main group.
657 mg/kg bw/day (570-821) in males; 781 mg/kg bw/ day (675-938) in females
Dose / conc.:
11 000 ppm
Remarks:
Recovery group.
657 mg/kg bw/day (570-821) in males; 781 mg/kg bw/ day (675-938) in females
No. of animals per sex per dose:
10 in main groups, 5 in recovery groups
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected based on results of a 28-day repeated dose toxicity study with oral exposure of Test Item in rats, results of an OECD 421 study in rats, and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Rationale for animal assignment: random
- Post-exposure recovery period in satellite groups: 5 weeks
Positive control:
Not required

Examinations

Observations and examinations performed and frequency:
MORTALITY/MURIBUNDITY CHEKS: Yes
- Time schedule: twice daily, in the morning and at the end of the working day.

CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
- Arena observations: before the first administration of the test item and then once weekly throughout the Dosing and Recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, starting on Day 1

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: weekly starting on Day 1

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): 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: end of treatment between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all animals
- Parameters checked in table 7.5.1/2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment between 7.00 and 10.30 a.m.
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all animals
- Parameters checked in table 7.5.1/2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: end of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all animals
- Parameters checked in table 7.5.1/2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during Week 12-13
- How many animals: 5 animals per sex per group
- Dose groups that were examined: Main animals
- Battery of functions tested:hearing ability / pupillary reflex / static righting reflex / fore- and hind-limb grip strength / locomotor activity

IMMUNOLOGY: Yes
- Time schedule for examinations: end of treatment
- How many animals: all males
- Dose groups that were examined: all groups
- Parameters examined: presence of alpha-2u globulin (hyaline droplet accumulation)
- Details: From the male animals of all Groups, additional slides (2-4 micrometers) of the kidney were prepared and stained for presence of alpha-2u globulin (hyaline droplet accumulation) using immunohistochemistry with antibody against rat alpha-2u globulin (R&D Systems, Minneapolis, USA). Biotin Goat Anti-Mouse Ig (BD Pharmingen, San Diego, USA) was used as secondary antibody, Streptavidin HRP (BD Pharmingen, San Diego, USA) was used as the detection enzyme and slides were stained with DAB-substrate (BD Pharmingen, San Diego, USA). A positive-, negative- and isotype control staining were performed on slides prepared from tissues of spare animals to verify staining was performed correctly. Tissues from the Main Study animals were preserved in 10% neutral buffered formalin for three weeks. Slides from the Main Study animals were stained with and without the use of two antigen retrieval steps, using Proteinase K and Citrate buffer. However, all staining attempts were not successful. Tissues from the Recovery Animals were preserved in 10% neutral buffered formalin for one day. Slides from the Recovery Animals were stained without antigen retrieval steps.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see "20153656 Tissues collection and preservation.pdf" in attached background document)

HISTOPATHOLOGY: Yes (see "20153656 Tissues collection and preservation.pdf" in attached background document)
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics; number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
6.1. Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
6.2. Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
6.3. Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Scabs and wounds were observed in individual animals which are commonly noted in rats of this age and strain, housed under laboratory conditions and/or were also seen in the control group, and were, at the incidence observed, not considered treatment-related.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No difference on body weight were observed in animals at 500 and 5000 ppm and body weight gain at 5000 ppm. An increase in body weight gain was observed in females at 500 ppm on several days, however it was considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
A decrease in body weight gain was observed for males from Day 29 and for females from Day 15 onwards at 11000 ppm. During the recovery period, body weights and body weight gain remained lower in Group 4 animals compared to control.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In males and females at 11000 ppm slightly lower absolute and relative food consumption was observed on Days 1-7, when compared to controls. Normal values for food consumption and relative food consumption were noted during the rest of the Dosing and Recovery Period.
Further statistically significant differences in food consumption for males were considered not to represent a change of toxicological significance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No changes were observed on haematological parameters at 500 ppm:
Mean corpuscular hemoglobin concentration was decreased in males at 5000 and 11000 ppm, this decrease was not observed after recovery.
At 11000 ppm in males, neutrophil numbers were slightly increased at end of treatment and platelet numbers at end of recovery when compared to control, however the alterations were considered unrelated to administration of the test item due to the minimal magnitude of the change.
Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Coagulation parameters of treated rats were considered not to have been affected by treatment. The statistically significant lower prothrombin time (PT) of males at 500 and 5000 ppm at the end of treatment was not considered to be of toxicological relevance as the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes were seen in females at 500 and 5000 ppm
At 500, 5000 and 11000 ppm increases in potassium levels were observed in males at the end of treatment. Potassium levels returned back to control values after the 5-week recovery period.
At 5000 and 11000 ppm an increase in total protein, albumin and cholesterol levels was noted in males and a decrease in chloride and creatinine levels was noted in females. Total protein, albumin, creatinine and chloride levels turned back to control values after the 5-week recovery period. Cholesterol levels in males at 11000 ppm were still increased after the recovery period.
At 11000 ppm an increase in calcium was observed in males, this increase was not observed at end of recovery. Moreover, in females, a decrease in albumin and total bilirubin levels was noted while an increase in cholesterol levels was noted. At the end of the 5-week recovery period, the decrease in albumin levels was still observed together with a decrease in total protein levels, however, these were considered to have arisen as a result of slightly high or control values and considered to be of no toxicological significance.
Increased Alanine aminotransferase and Aspartate aminotransferase activity were noted for one female animal at 11000 ppm. In absence of any histopathological correlation, and as these findings were not observed in the other animals of the same group, these findings were considered to be not toxicological relevant.
Any other statistically significant changes in clinical chemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males at 500, 5000 and 11000 ppm and in females at 11000 ppm a decrease in chloride levels was observed at the end of treatment. At 5000 and 11000 ppm, a decrease in creatinine and potassium levels was observed in females at the end of treatment, however these changes were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend. At the end of the 5-week recovery period, these parameters were similar between animals treated at 11000 ppm and controls.
Values in treated animals achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high or low control values and in the absence of a treatment-related distribution and considered to be of no toxicological significance.
All other urine parameters of treated rats were considered not to have been affected by treatment.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity were 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 weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related organ weight differences were noted in the liver of both sexes, kidney of males, and adrenal gland of females, as summarized in Table 7.5.1/3 and Table 7.5.1/4.
Higher liver weights were noted in males and females at 11000 ppm (absolute and relative to body weight) and in males at 5000 ppm (relative to body weight only). At 11000 ppm this correlated with minimal centrilobular hypertrophy in both sexes. After the recovery period at 11000 ppm higher liver weights were noted only in males, only relative to body weight and at a lower magnitude than the end of treatment, consistent with a partial recovery. No liver weight differences were noted in recovery females, consistent with a complete recovery.
At the end of treatment, higher kidney weights were noted in males only starting at 5000 ppm (relative to body weight only) and 11000 ppm (absolute and relative to body weight) with a dose-related increase in magnitude. This correlated histologically with test item-related microscopic changes including primarily increased incidence and severity of hyaline droplet accumulation, and to a lesser degree, presence of granular casts and increased inflammatory cell infiltrates. Following the recovery period at 11000 ppm, higher kidney weights were noted relative to body weight only and at a lower magnitude than the end of treatment, consistent with a partial recovery.
Higher adrenal gland weights were noted in females only at the end of treatment at 11000 ppm (absolute and relative to body weight). There was no histologic correlate. Following the recovery period at 11000 ppm, differences in adrenal gland weights were not noted, consistent with a complete recovery.
Any other differences, including those that reached statistical significance, were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with the Test Item were noted in the kidney and thyroid gland of males only, and in the liver of both sexes. These findings are summarized in Table 7.5.1/5 and Table 7.5.1/6.
In the kidney, in males only, microscopic findings were noted starting at 500 ppm without a clear dose relationship. This included a combination of hyaline droplet accumulation (increased incidence and severity, up to marked), granular casts (up to moderate), basophilic tubules (increased incidence and severity, up to marked), and inflammatory cell infiltrates (increased incidence and severity, up to slight). These findings correlated with higher kidney weights. Following the recovery period hyaline droplet accumulation and inflammatory cell infiltrates were comparable to the concurrent control males, consistent with full recovery. Granular casts and increased incidence and severity of tubular basophilia wereas present, although at slightly reduced incidence and severity compared to the end of treatment and marginally greater than the recovery control group, consistent with partial recovery. However, it is noted that a 5-week recovery period may not be sufficient time to observe recovery of such effects because of the slow rate of turnover of tubular epithelial cells in the rat kidney. In the recovery males, positive staining for alpha-2u globulin via immunohistochemistry was present in all animals and was comparable between the control and treated groups.
In the liver, minimal centrilobular hypertrophy was noted of males at 11000 ppm and females at 5000 and 11000 ppm. This microscopic finding correlated with higher liver weights. Following the recovery period, hypertrophy was not noted in females at 11000 ppm (recovered), but was seen in males at 11000 ppm, and was generally of comparable incidence and severity as the end of the main study (not recovered). This correlated with higher liver weights in males (relative to body weight only).
In the thyroid gland, in males only, diffuse bilateral, follicular cell hypertrophy was noted at 11000 ppm at higher severity (up to slight) compared to the control group. Following the recovery period, the only minimal follicular cell hypertrophy was noted in males at 11000 ppm and the incidence was comparable to the concurrent controls (recovered).
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
One renal tubular adenoma was noted in a high dose recovery female. This is a well-known spontaneous tumor in Sprague-Dawley rats (amphophilic-vacuolar tubular adenoma, Crabbs et al., 2013) and in the absence of any test item-related changes in the kidney of main study females was considered to be spontaneous and not related to the test item.
Other effects:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
11 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
11 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: male rat-specific effects observed from 500 ppm in the kidneys of male; not relevant to human

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 7.5.1/3: Mean Percent Liver and Kidney Weight Differences from Control Groups (Males)

 

Main Males

Recovery Males

Dose level (ppm)

500

5000

11000

11000

 

 

 

 

 

LIVER

 

 

 

 

              Absolute

6.0

13.9

23.7**

3.7

              Relative to body weight

11.1

18.7**

31.1**

12.1*

 

 

 

 

 

KIDNEYS

 

 

 

 

              Absolute

9.8

15.5

19.7*

9.7

              Relative to body weight

13.8

18.5*

26.2**

18.0**

*: P<0.05, **: P<0.01

 

 

 

 

 

 

 

 

Table 7.5.1/4: Mean Percent Liver and Adrenal Gland Weight Differences from Control Groups (Females)

 

Main Females

Recovery Females

Dose level (ppm)

500

5000

11000

11000

 

 

 

 

 

LIVER

 

 

 

 

              Absolute

10.4

11.0

21.4*

-8.3

              Relative to body weight

8.4

11.9

24.9**

0.0

 

 

 

 

 

ADRENAL GLAND

 

 

 

 

              Absolute

1.5

12.3

16.9*

-15.1

              Relative to body weight

0.0

13.6

18.2*

-9.1

*: P<0.05, **: P<0.01

 

 

 

 

 

Table 7.5.1/5: Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals (Males)

 

Main Males

Recovery Males

Dose level (ppm)

0

500

5000

11000

0

11000

Group Number

1

2

3

4

1

4

Necropsy Day

 

 

 

 

 

 

 

 

 

 

 

 

 

KIDNEYSa

10

10

10

10

5

5

Hyaline droplet accumulation

2

10

10

10

4

1

Minimal

2

-

-

2

4

1

Slight

-

2

3

2

-

-

Moderate

-

7

7

4

-

-

Marked

-

1

-

2

-

-

 

 

 

 

 

 

 

Casts, granular

0

2

7

5

0

2

Minimal

-

2

2

-

-

-

Slight

-

-

5

4

-

1

Moderate

-

-

-

1

-

1

 

 

 

 

 

 

 

Tubular basophilia

4

10

10

9

4

4

Minimal

3

-

-

2

3

2

Slight

1

6

3

2

1

1

Moderate

-

3

6

5

-

1

Marked

-

1

1

-

-

-

 

 

 

 

 

 

 

   Infiltrate, inflammatory cell

3

6

6

7

2

2

      Minimal

3

5

3

6

2

2

      Slight

-

1

3

1

-

-

 

 

 

 

 

 

 

LIVERa

10

10

10

10

5

5

    Hypertrophy, hepatocellular

0

0

0

2

0

2

      Minimal

-

-

-

2

-

2

 

 

 

 

 

 

 

THYROID GLANDa

10

10

10

10

5

5

    Hypertrophy, follicular

4

6

3

5

3

2

      Minimal

4

6

3

3

3

2

      Slight

-

-

-

2

-

-

a = Number of tissues examined from each group.

Table 7.5.1/6: Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals (Females)

 

Main Females

Recovery Females

Dose level (ppm)

0

500

5000

11000

0

11000

Group Number

1

2

3

4

1

4

Necropsy Day

 

 

 

 

 

 

 

 

 

 

 

 

 

LIVERa

10

10

10

10

5

5

    Hypertrophy, hepatocellular

0

0

2

6

0

0

      Minimal

-

-

2

6

-

-

a = Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:
Based on the results for the kidney, the no-observed-adverse-effect level (NOAEL) was considered to be below 500 ppm for male rats and at least 11000 ppm for females. The male rat-specific nature of the effects seen in the kidney are considered not relevant to human therefore the NOAEL was considered to be at least 11000 ppm for human (corresponding to 657 mg/kg bw/day for males and 781 mg/kg bw/day for females).
Executive summary:

In a subchronic toxicity study performed according to the OECD TG No. 408 and in compliance with GLS,Sprague Dawley rats were treated with the Test Item for 90 consecutive days by dietary administration at dose levels of 0, 500, 5000 and 11000 ppm (corresponding to 29, 287 and 657 mg/kg bw/day for males and 37, 351 and 781 mg/kg bw/day for females). Reversibility was assessed with a 5-week treatment-free recovery period for Control and high-dose animals.

The study design was as follows:

 

Group No.

Test Item Id.

Dose Level

(ppm)

Subsets

Number of Animals

Males

Females

1

Test Item

0 (Control)

Main

10

10

Recovery

5

5

2

500

Main

10

10

3

5000

Main

10

10

4

11000

Main

10

10

Recovery

5

5

 

Chemical analyses of dietary preparations were conducted three times during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, ophthalmology, functional tests, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), gross necropsy findings, organ weights, and histopathologic examinations.

Sprague Dawley rats were treated with Test Item for 90 consecutive days by dietary administration at dose levels of 0, 500, 5000 and 11000 ppm, followed by a 5-week treatment-free recovery period.

Test dietswere considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. Test dietswere consideredstable, for at least three weeks in the freezer (≤ -15°C) and 5 days at room temperature.

At 11000 ppm, a slight decrease in body weight gain was observed in males and females with correlating lower (relative) food consumption. These changes were considered to be test item-related but non-adverse.
Clinical pathology analyses at 11000 ppm showed test item-related decreased mean corpuscular haemoglobin concentration, increasedtotal protein, albumin, cholesterol, calcium and potassium levels in males and decreased creatinine and chloride levels in females.At the severity observed, and in the absence of any histopathological correlation, these effects were considered not to be adverse.
Test item-related microscopic findings were present in the kidney, liver and thyroid gland at 11000 ppm. In males in the kidneythere was increased incidence/severity of hyaline droplet accumulation, inflammatory cell infiltrates and tubular basophilia and presence of granular casts at the end of treatment. In Recovery males at 11000 ppm, the incidence and severity of hyaline droplet accumulation and inflammatory cell infiltrates were comparable to the concurrent control males, but granular casts and an increased incidence and severity of tubular basophilia were still present at the end of the Recovery phase. Positive staining for alpha-2u globulin via immunohistochemistry was present in all Recovery animals and was comparable between the control and treated groups. Kidney findings were considered adverse to the male rat due to the presence of degenerative changes (granular casts and high severity of tubular basophilia).
At 11000 ppm, in the liver of males and females there was minimal centrilobular hypertrophy in main study animals, which showed recovery for females only, which were considered to be non-adverse in both sexes. 
In the thyroid gland, there was an increased severity of diffuse follicular cell hypertrophy in main study males compared to control males, which showed complete recovery after the treatment-free period.
A remaining finding was a higher adrenal gland weight in females at 11000 ppm, with complete recovery. At the severity observed and in absence of a histopathological correlation, this finding was considered not to be adverse. 

At 5000 ppm test item related clinical pathology findings were observed including a decrease inmean corpuscular hemoglobin concentration, anincrease in potassium, total protein, albumin and cholesterol levelsin malesand a decrease in chloride and creatinine levels in females. At the severity observed and in the absence of any histopathological correlation, these effects were considered not to be adverse.
Moreover, test item-related microscopic findings were present in the kidney of the males, including increased incidence/severity of hyaline droplet accumulation, inflammatory cell infiltrates and tubular basophilia and presence of granular casts at the end of the main study and were considered to be adverse to the male rat. Females at this dose showed minimal centrilobular hypertrophy of the liver, which were considered to be non-adverse.

At 500 ppm similar adverse test item-related microscopic findings were observed in the kidneys of the males without a clear dose relationship. Moreover, clinical pathology analyses showed increased potassium levels in the males. At the severity observed and in the absence of any histopathological correlation, this effect was considered not to be adverse.
No toxicologically significant changes were noted in the females at 500 ppm. 

 

No treatment-related toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations, ophthalmoscopy, coagulation and macroscopic examination).

In conclusion, administration of the Test Item by dietary administration resulted in decreased body weight gain and food consumption in both males and females at 11000 ppm. Effects were observed in males at 500, 5000 and 11000 ppm and consisted of higher kidney weight, increased incidence/severity of hyaline droplet accumulation, inflammatory cell and tubular basophilia and presence of granular casts consistent with ‘hyaline droplet nephropathy’. After recovery, hyaline droplet accumulation was comparable in control and treated animals, but granular casts and increased incidence/severity of tubular basophilia showed partial recoveryindicating the potential for reversibility of effects but which remained incomplete in the short recovery period. Non-adverse test item-related changes were noted in the liver of males and females starting at 5000 ppm (minimal centrilobular hypertrophy and/or higher weight), thyroid gland of males at 11000 ppm (increased severity of follicular cell hypertrophy), and adrenal gland of females at 11000 ppm (higher weight). These non-adverse findings were not noted after the recovery period, with the exception of minimal centrilobular hypertrophy and higher liver weight in males at 11000 ppm. Based on the results for the kidney, the no-observed-adverse-effect level (NOAEL) was considered to be below 500 ppm for male rats and at least 11000 ppm for females. The male rat-specific nature of the effects seen in the kidney are considered not relevant to human, therefore the NOAEL was considered to be at least 11000 ppm for human (corresponding to 657 mg/kg bw/day for males and 781 mg/kg bw/day for females).

 

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