Registration Dossier

Administrative data

Description of key information

In the 90-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for systemic toxicity was 65 mg/kg bw/day based on test substance-related findings of bile duct hyperplasia in the liver and chronic progressive nephropathy in the kidney with corresponding higher liver and kidney weights at 300 mg/kg bw/day (Charles River, 2020).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17th of June 2019 to 07 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks old.
- Weight at study initiation: between 161 and 288 g.
- Fasting period before study: No
- Housing: Polycarbonate, solid-bottom cages containing Bed-O-Cobs® or other bedding material.
- Diet: LLC Certified Rodent LabDiet 5002, ad libitum
- Water: Municipal tap water, treated by reverse osmosis and ultraviolet irradiation, ad libitum
- Acclimation period: At least 7 days

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants of the food as well as periodic analysis of the water were provided by the supplier and are on file at the Testing Facility. It was considered that no known contaminants in the feed or water were present that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 26°C
- Humidity (%): 30% to 70%
- Air changes (per hr): ≥10
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: May 2019 To: 20/21 November 2019.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing formulations were prepared at least weekly at appropriate concentrations to meet the dose level requirements.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on the test substance’s characteristics and the subsequent relevant OECD testing guidelines.
- Concentration in vehicle: 0, 3.75, 16.25, 75 mg/mL
- Amount of vehicle: 0, 3.75, 16.25, 75 mg/mL
- Lot/batch no.: 2IC0148, 1IG1538, 2IH0387
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected for concentration analysis at the first and last sample preparation. Samples for homogeneity were collected at the same intervals, however, from the low and top doses only. All samples were analysed the same day where possible, alternatively stored in a refrigerator, set to maintain a target of 5°C until analysis.
Duration of treatment / exposure:
90-day exposure
Frequency of treatment:
Daily for 90 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
65 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals per sex per dose. For the recovery group, 5 additional animals per sex for the control and 600 mg/kg bw/day dose groups were used, respectively.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The target exposure concentrations were selected by the sponsor representative in consultation with the study director based on a previous Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD Test Guideline 422) where the administrated doses were 0, 50, 150 and 600 mg/kg bw/day. In the dose group of 600 mg/kw bw/day, lowered body weights were observed in both males and females. Additionally, microscopically changes occurred in the liver in the 150 and 600 mg/kg bw/day and correlated with higher mean absolute and relative liver weights as well as altered clinical biochemistry parameters in the 600 mg/kg bw/day dose group. The doses considered to cause adverse effects were 600 mg/kg bw/day for males and 150 mg/kg bw/day for females.
Based on these findings, dose levels of 15, 65, and 300 mg/kg/day were selected for this study with the aim to induce toxic effects at the highest dose, however, not death or severe suffering.
- Rationale for animal assignment: randomised scheme designed to achieve similar group mean body weights (within ± 20% of the mean for each).
- Fasting period before blood sampling for clinical biochemistry: yes, at least 8 hours, however, no more than 24 hours.
- Rationale for selecting satellite groups: randomised
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale: randomised
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-side observations were made at 0.5 - 3 hours post-dosing and at least once daily on non-dosing/recovery days. Cage side observations were not conducted on days when the detailed clinical observations were performed during the non-dosing and/or recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made outside the home cage in a standard arena once before the first administration, once a week (± 2 days) thereafter during the study period and on the day of scheduled necropsy (for animals selected for necropsy). The observations focused on posture, convulsions/tremors, biting, faeces consistency, lacrimation/chromodacryorrhoea, salivation, piloerection, fur appearance, palpebral closure, respiratory rate/character, red/crusty deposits, mucous membranes/eye/skin colour, eye prominence and muscle tone.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the first administration, once a week (± 2 days) thereafter during the study period and on the day of scheduled necropsy (for animals selected for necropsy).

FOOD CONSUMPTION: Yes
Food consumption was measured weekly (± 2 days) throughout the study period.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during the pre-treatment period (included unused replacement animals) and once at the end of the dosing period (day 89).
- Dose groups that were examined: All main study and recovery animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, at least 8 hours, however, no more than 24 hours.
- How many animals: all animals scheduled for necropsy.
- Parameters checked in table were examined: yes, see table 1.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy.
- Animals fasted: Yes, for at least 8 hours, however, not more than 24 hours.
- How many animals: all animals
- Parameters checked in table were examined: yes, see table 1.

URINALYSIS: Yes
- Time schedule for collection of urine: On the day of scheduled necropsy.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for at least 8 hours, however, not more than 24 hours.
- Parameters checked in table were examined: Yes, see table 1.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the final week of the test substance administration and near the end of the recovery period.
- Dose groups that were examined: all main study and recovery animals.
- Battery of functions tested: Functional observational battery (FOB) assessment was performed and the following parameters were observed: sensory observations including approach response, touch response, startle response, tail pinch response, olfactory orientation, pupil response, eyeblink response, forelimb extension, hindlimb extension, air righting reflex. Neuromuscular observations included hindlimb extensor strenght, grip-strenght-hind and forelimb, rotarod performance and hindlimb foot splay.

IMMUNOLOGY: No

OTHER: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)

HISTOPATHOLOGY: Yes (see table 2)
Other examinations:
Hormone sample analysis: TSH, T3 and T4 hormone analysis were performed.
Statistics:
Means, standard deviations, ratio, percentages, numbers, and/or incidences were reported as appropriate by dataset. All statistical analyses were performed within the respective study phase. Moreover, all statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1% and 5% levels, unless otherwise noted. Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance related clinical observations. All clinical observations in the test substance treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose related manner, and/or were common findings for laboratory rats of this age and strain.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test substance-related deaths. However, 1 male from the 300 mg/kg bw/day group and 1 male from the 15 mg/kg bw/day group were found dead during the dosing period. Histopathology revealed that the cause of death of the male from 300 mg/kg bw/day was inflammation in the kidney, while the cause of death for the second male at 15 mg/kg bw/day undetermined. All other animals survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related effects on body weights and/or body weight gains were noted in the 15, 65, and 300 mg/kg bw/day group males, compared to the control group, for the duration of the dosing period. A statistically significantly lower cumulative body weight gain during the dosing period was noted in 300 mg/kg bw/day group males compared to the control group. At the end of the dosing period, the body weights for the 15, 65, and 300 mg/kg bw/day group males were 2.0%, 2.9%, and 8.0% lower than the control group. During the recovery period, the 300 mg/kg bw/day group males recovered, as noted by body weight gains that were comparable to or higher than the gains in the control group; however, at the end of the recovery period, the body weight in the 300 mg/kg bw/day group males remained lower compared to the control group. Recovery was not assessed for the 15 and 65 mg/kg bw/day groups.
There were no test substance-related effects on body weight or body weight gains noted in the female groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test substance-related effects on food consumption. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. These differences in food consumption were attributed to biological variability and were not considered related to test article administration due to lack of a dose-response trend.
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 ophthalmic lesions indicative of toxicity were observed in any of the test substance treated groups.
Haematological findings:
no effects observed
Description (incidence and severity):
Hematology parameters were unaffected by test substance administration at the terminal and recovery necropsy. Coagulation parameters were unaffected by test substance administration.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related effects on clinical chemistry parameters in the 300 mg/kg bw/day group males at the end of the dosing period included dose-related lower mean levels of triglycerides compared to the control group. At the end of the recovery period, there were no test substance related clinical chemistry changes noted in the 300 mg/kg bw/day group males.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis parameters were unaffected by test substance administration.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No effects were observed in any of the test substance treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Terminal euthanasia: Test substance-related higher liver and kidney weights were noted in the 300 mg/kg bw/day group males and females at the terminal euthanasia. Organ weight changes were not observed at the recovery euthanasia.
Higher mean liver weights correlated with histopathologic findings of hepatocellular hypertrophy and bile duct hyperplasia in the 300 mg/kg bw/day group males and females. There were no histopathologic correlations for the higher mean kidney weights.
No other test substance-related organ weight changes were noted.

Recovery euthanasia: Test substance-related organ weight changes noted at the terminal euthanasia were not observed at the end of the recovery period (Day 120/121). No other test substance-related organ weight changes were noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related gross findings were noted at terminal euthanasia or recovery euthanasia.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
TERMINAL EUTHANASIA
Test substance-related microscopic findings were noted in the liver, kidney, and thyroid gland in the 65 and 300 mg/kg bw/day group males and in the liver and thyroid gland in the 300 mg/kg bw/day females.
LIVER: In the liver, hepatocellular hypertrophy was noted in the 65 and 300 mg/kg bw/day group males and the 300 mg/kg bw/day group females and characterised by enlarged hepatocytes with a homogenous, eosinophilic cytoplasm which predominantly affected centrilobular hepatocytes. Bile duct hyperplasia was noted in the 300 mg/kg bw/day group males and females and characterised by increased numbers of bile duct profiles in the portal areas. Bile duct hyperplasia was accompanied by peribiliary fibrosis in the 300 mg/kg bw/day group males and characterised by increased fibrous connective tissue arranged in concentric layers around portal areas. Pigment was noted in the 300 mg/kg bw/day group males and females and characterised by the presence of a dark brown substance in the bile ducts which under polarized light exhibited red birefringence and rare “Maltese Cross”-like structures. Histopathologic findings of hepatocellular hypertrophy and bile duct hyperplasia in the liver correlated with higher mean liver weights in the 300 mg/kg bw/day group males and females. Hepatocellular hypertrophy was considered adaptive and non-adverse. Bile duct hyperplasia was considered adverse at 300 mmg/kg bw/day, based on severity.
KIDNEY: In the kidney, an increased incidence and severity of chronic progressive nephropathy (CPN), characterised by the presence of basophilic tubular epithelial cells and thickened basement membrane with occasional mononuclear infiltrates, was noted in the 65 and 300 mg/kg bw/day group males. The finding of CPN was considered adverse at 300 mg/kg bw/day and non-adverse at 65 mg/kg bw/day based on severity, but was of no known toxicologic importance as this finding has no human counterpart and no relevance for extrapolation in human risk assessment (Hard et al., 2009). The nature of the higher mean weights in the kidneys was undetermined as there were no histopathologic correlates and the histopathologic findings observed likely did not appreciably contribute to kidney weight.
An increased incidence of minimal tubular mineralization in the kidney was noted in the 300 mg/kg bw/day group females, characterised by mineralized intraluminal material in the proximal convoluted tubules at the corticomedullary junction. This is a common background finding in rats (McInnes, 2012) and was not considered test substance-related.
THYROID GLAND: In the thyroid gland, epithelial hypertrophy was noted in the 65 and 300 mg/kg bw/day group males and the 300 mg/kg bw/day group females and characterised by the increase in size and height of epithelial cells lining the follicles and a decrease in the size of follicular lumina. Colloid alteration was also noted in the 300 mg/kg bw/day group males and characterised by clumped, basophilic colloid within the lumina. These findings were not considered adverse based on severity.
STOMACH: In the stomach, basal cell hyperplasia was noted in 1 female (No. 4505) from the 300 mg/kg bw/day group and characterised by the presence of an isolated nest of basal cells in the lamina propria at the junction of the nonglandular and glandular stomach. The nest of cells had an intact basement membrane and there was no evidence of dysplasia. In the same female (No. 4505), squamous metaplasia was noted in the rectum and characterised by focal replacement of normal epithelium by squamous epithelium. Neither finding has been recorded as an incidental lesion in the Charles River Ashland historical control database. However, examination of the gastrointestinal tract from the stomach to the rectum from all remaining animals in this study did not reveal any proliferative or metaplastic lesions. Therefore, the relationship of these findings in the stomach and rectum of animal No. 4505 was uncertain.

RECOVERY EUTHANASIA
LIVER: At the recovery necropsy, test substance-related bile duct hyperplasia, peribiliary fibrosis, and pigment were observed in the livers of the 300 mg/kg bw/day group males. The severity of the test substance-related findings were trending lower at the recovery euthanasia.
KIDNEYS: Minimal chronic progressive nephropathy was still observed in the kidneys of the 300 mg/kg bw/day group males (2 out of 5 animals vs 1 out of 5 animals from the concurrent control group males) but was of similar incidence and severity as observed in the Charles River Ashland historical control database. Therefore, test substance-related chronic progressive nephropathy observed in the 300 mg/kg bw/day group males at the terminal euthanasia was considered fully recovered at the recovery euthanasia.

The remaining test substance-related findings noted at the terminal euthanasia were not observed at the end of the recovery period (Day 120/121). Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of Sprague Dawley rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A finding of adenocarcinoma, consistent with mammary adenocarcinoma, was observed in the skin of 1 female from the 300 mg/kg bw/day group. Mammary gland adenocarcinomas have been observed as an incidental lesion with an incidence rate of up to 18.64% in the Charles River Ashland historical control database and, although rare, have been reported as incidental findings in young Sprague Dawley rats (Ikezaki et al., 2011; Weber, 2017). Additionally, there were no test substance-related findings observed in the mammary glands of the 300 mg/kg bw/day group females at the terminal euthanasia. Therefore, the adenocarcinoma observed in the skin was not considered test substance-related.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related effects on thyroid hormone parameters were noted in the 65 mg/kg bw/day group females and 300 mg/kg bw/day group male and females. The 300 mg/kg bw/day group males had lower T4 levels at the terminal necropsy relative to the control group, coupled with thyroid gland hypertrophy observed microscopically. T3 levels were lower in a dose-responsive manner in the 65 and 300 mg/kg bw/day group females relative to the control group. TSH levels were unaffected by the administration of the test substance. There were no differences in any thyroid hormone parameter at the recovery necropsy; recovery was only assessed at 300 mg/kg bw/day.
Details on results:
The number of females in estrus, diestrus, or proestrus were comparable between the control and test substance-treated groups at the terminal and recovery euthanasias.
Key result
Dose descriptor:
NOAEL
Effect level:
65 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
65 mg/kg bw/day (actual dose received)
System:
other: hepatobiliary and urinary
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 3: Summary of organ weight data at terminal euthanasia (Day 91/92)a

 

Males

Males

Males

Females

Females

Females

Group

2

3

4

2

3

4

Dose (mg/kg/day)

15

65

300

15

65

300

No. Animals per Group

9

10

9

10

9

10

Liver (No. Weighed)b

(9)

(10)

(9)

(10)

(9)

(10)

    Absolute value

3.13

4.67

26.92*

3.49

3.97

18.11*

    % of body weight

4.34

7.38

39.73*

1.24

7.80

26.62*

    % of brain weight

0.17

2.77

26.51*

1.81

6.03

19.89*

Kidney (No. Weighed)b

(9)

(10)

(9)

(10)

(9)

(10)

    Absolute value

5.96

7.32

22.46*

10.26

4.38

16.75*

    % of body weight

7.57

9.92

34.13*

8.32

8.21

25.07*

    % of brain weight

2.89

5.33

22.04*

8.40

6.43

18.52*

a  Organ weight values rounded to 2 decimal places (values with all decimal places in Provantis tables).

b All values expressed as percent difference of control group means.

Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group – P< 0.05.

Bold and asterisk (*) are significantly different from control group – P<0.01

Table 4: Summary of Microscopic Findings – Terminal Euthanasia (Day 91/92)

 

Males

Males

Males

Males

Females

Females

Females

Females

Group

1

2

3

4

1

2

3

4

Dose (mg/kg/day)

0

15

65

300

0

15

65

300

No. Animals per Group

10

9

10

9

10

10

9

10

Liver (No. Examined)

(10)

(9)

(10)

(9)

(10)

(10)

(9)

(10)

Hypertrophy, hepatocellular

0

0

1

5

0

0

0

9

     Minimal

-

-

1

5

-

-

-

9

Hyperplasia, bile duct

0

0

0

7

0

0

0

2

     Minimal

-

-

-

3

-

-

-

2

     Mild

-

-

-

2

-

-

-

0

     Moderate

-

-

-

2

-

-

-

0

Fibrosis, peribiliary

0

0

0

4

0

0

0

0

     Minimal

-

-

-

2

-

-

-

-

     Mild

-

-

-

2

-

-

-

-

Pigment

0

0

0

8

0

0

0

1

     Minimal

-

-

-

4

-

-

-

1

     Mild

-

-

-

3

-

-

-

0

     Moderate

-

-

-

1

-

-

-

0

Kidney (No. Examined)

(10)

(9)

(10)

(9)

(10)

(0)

(0)

(10)

Chronic progressive nephropathy

5

5

7

7

0

N/A

N/A

0

     Minimal

5

4

6

4

-

N/A

N/A

-

     Mild

0

1

1

2

-

N/A

N/A

-

     Moderate

0

0

0

1

-

N/A

N/A

-

Thyroid gland (No. Examined)

(10)

(9)

(10)

(9)

(10)

(10)

(9)

(10)

Hypertrophy, epithelial

0

0

2

7

0

0

0

5

     Minimal

-

-

2

6

-

-

-

5

     Mild

-

-

0

1

-

-

-

0

Colloid alteration

0

0

0

3

0

0

0

0

     Minimal

-

-

-

3

-

-

-

-

- = No noteworthy findings.

N/A = not applicable.

Table 5: Summary of Microscopic Findings – Recovery Euthanasia (Day 120/121)

 

Males

Males

Males

Males

Females

Females

Females

Females

Group

1

2

3

4

1

2

3

4

Dose (mg/kg/day)

0

15

65

300

0

15

65

300

No. Animals per Group

5

N/A

N/A

5

5

N/A

N/A

5

Liver (No. Examined)

(5)

N/A

N/A

(5)

(5)

N/A

N/A

(5)

Hyperplasia, bile duct

0

N/A

N/A

4

0

N/A

N/A

0

     Minimal

-

N/A

N/A

3

-

N/A

N/A

-

     Mild

-

N/A

N/A

1

-

N/A

N/A

-

Fibrosis, peribiliary

0

N/A

N/A

1

0

N/A

N/A

0

     Mild

-

N/A

N/A

1

-

N/A

N/A

-

Pigment

0

N/A

N/A

4

0

N/A

N/A

0

     Minimal

-

N/A

N/A

3

-

N/A

N/A

-

     Mild

-

N/A

N/A

1

-

N/A

N/A

-

- = No noteworthy findings.

N/A = not applicable.

References:

Hard GC, Johnson KJ, Cohen SM. A comparison of rat chronic progressive nephropathy with human renal disease-implications for human risk assessment. Crit Rev Toxicol. 2009;39(4):332 346

Ikezaki S, Takagi M, Tamura K. Natural occurrence of neoplastic lesions in young Sprague dawley rats. Toxicol Pathol. 2011;24(1):37-40.

McInnes EF. Wistar and Sprague-Dawley Rats. In: Background lesions in Laboratory Animals, A Color Atlas. Edinburgh: Saunders Elsevier; 2012:35.

Weber K. Differences in Types and Incidence of Neoplasms in Wistar Han and Sprague-Dawley Rats. Toxicol Pathol. 2017;45(1):64-75.

Conclusions:
In the 90-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP, the concluded NOAEL for systemic toxicity was 65 mg/kg bw/day based on test substance-related findings of bile duct hyperplasia in the liver and chronic progressive nephropathy in the kidney with corresponding higher liver and kidney weights at 300 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
65 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
other: hepatobiliary and urinary
Organ:
kidney
liver

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

In the 90-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP, 10 male and 10 female rats per group were administered daily oral (gavage) doses of 0, 15, 65 and 300 mg/kg bw/day 1,1,3,3-tetramethyl-1,3-divinyldisiloxane in corn oil for 90 days. There were 5 additional male and female rats to the control and high dose group, which served as recovery group and were observed for additional 28 days.

The following parameters and end points were evaluated in the study: clinical signs, body weights, body weight gains, food consumption, functional observational battery, motor activity, ophthalmology, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), hormone analyses (T3, T4, and TSH), estrous cycle, gross necropsy findings, organ weights, and histopathologic examinations.

There were no test substance-related deaths. However, one male from the 300 mg/kg bw/day group and 1 male from the 15 mg/kg bw/day group were found dead during the dosing period. Histopathology revealed that the cause of death of the male from 300 mg/kg bw/day was inflammation in the kidney, while the cause of death for the second male at 15 mg/kg bw/day was undetermined. All other animals survived to the scheduled necropsies.

There were no test substance-related clinical observations or effects on food consumption, functional observational battery, motor activity, hematology coagulation, urinalysis, or estrous cycle. There were no test substance related ophthalmic or gross necropsy findings.

Test substance-related effects on body weights and/or body weight gains were noted in the 15, 65, and 300 mg/kg bw/day group males, compared to the control group, for the duration of the dosing period. A statistically significantly lower cumulative body weight gain during the dosing period was noted in 300 mg/kg bw/day group males compared to the control group. At the end of the dosing period, the body weights for the 15, 65, and 300 mg/kg bw/day group males were 2.0%, 2.9%, and 8.0% lower than the control group. During the recovery period, the 300 mg/kg bw/day group males recovered, as noted by body weight gains that were comparable to or higher than the gains in the control group; however, at the end of the recovery period, the body weight in the 300 mg/kg bw/day group males remained lower compared to the control group. Recovery was not assessed for the 15 and 65 mg/kg bw/day groups.

Test substance-related effects on clinical chemistry parameters in the 300 mg/kg bw/day group males at the end of the dosing period included dose-related lower mean levels of triglycerides compared to the control group. At the end of the recovery period, there were no test substance related clinical chemistry changes noted in the 300 mg/kg bw/day group males.

Test substance-related effects on thyroid hormone parameters were noted in the 65 mg/kg bw/day group females and 300 mg/kg bw/day group male and females. The 300 mg/kg bw/day group males had lower T4 levels at the terminal necropsy relative to the control group, coupled with thyroid gland hypertrophy observed microscopically. T3 levels were lower in a dose-responsive manner in the 65 and 300 mg/kg bw/day group females relative to the control group. Thyroid stimulating hormone (TSH) levels were unaffected by the administration of the test substance. There were no differences in any thyroid hormone parameter at the recovery necropsy; recovery was only assessed at 300 mg/kg bw/day.

Test substance-related higher liver and kidney weights were noted in the 300 mg/kg bw/day group males and females at the terminal euthanasia. Organ weight changes were not observed at the recovery euthanasia.

Test substance-related microscopic findings were noted in the liver, kidney, and thyroid gland in the 65 and 300 mg/kg bw/day group males and in the liver and thyroid gland in the 300 mg/kg bw/day females. In the liver, hepatocellular hypertrophy was noted in the 65 and 300 mg/kg bw/day group males and the 300 mg/kg bw/day group females. Bile duct hyperplasia was noted in the 300 mg/kg/day group males and females. Bile duct hyperplasia was accompanied by peribiliary fibrosis in the 300 mg/kg/day group males. Pigment was noted in the 300 mg/kg bw/day group males and females. Histopathologic findings of hepatocellular hypertrophy and bile duct hyperplasia in the liver correlated with higher liver weights in the 300 mg/kg bw/day group males and females. Hepatocellular hypertrophy was considered adaptive and non-adverse. Bile duct hyperplasia was considered adverse at 300 mg/kg bw/day, based on severity. In the kidney, an increased incidence and severity of chronic progressive nephropathy was noted in the 65 and 300 mg/kg bw/day group males; this finding was considered adverse at 300 mg/kg bw/day based on severity but was of no known toxicologic importance as this finding has no human counterpart and no relevance for extrapolation in human risk assessment (Hard et al., 2009). In the thyroid gland, epithelial hypertrophy was noted in the 65 and 300 mg/kg bw/day group males and the 300 mg/kg bw/day group females. Colloid alteration was also noted in the 300 mg/kg bw/day group males. These findings were not considered adverse based on severity.

In conclusion, the NOAEL for systemic toxicity was concluded to be 65 mg/kg bw/day based on test substance-related findings of bile duct hyperplasia in the liver and chronic progressive nephropathy in the kidney with corresponding higher liver and kidney weights at 300 mg/kg bw/day. Nonadverse, test substance-related microscopic findings were noted in the liver, kidney, and thyroid gland at 65 and 300 mg/kg bw/day. Test substance-related histologic findings and organ weight changes observed at the terminal euthanasia were no longer observed at the recovery euthanasia.

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test followed by a 14-day recovery period (WIL, 2011), conducted according to OECD Test Guideline 422 and in compliance with GLP, oral administration of 1,1,3,3-tetramethyl-1,3-divinyldisiloxane was well tolerated at doses of 50, 150 or 600 mg/kg bw/day. Clinical findings considered to be attributable to the treatment included clear material around the mouth recorded in a dose-related manner in the 150 and 600 mg/kg bw/day groups, red material around the mouth in the 600 mg/kg bw/day group at approximately 1 hour following dose administration, and salivation prior to dosing for females in this group. Test substance-related mean body weight losses or reduced mean body weight gains were generally noted for males and females throughout the treatment period and a corresponding reduction in mean food consumption was noted for high dose males during the first week of treatment. Mean body weights, body weight gains, and food consumption were unaffected by treatment in the 50 and 150 mg/kg bw/day groups. Increases in mean cumulative total and ambulatory counts were noted for males in the 600 mg/kg bw/day group due to slower habituation over the test session. No effects were noted on locomotor activity for males at 50 and 150 mg/kg bw/day or females at all dosage levels or on FOB parameters for males and females at all dosage levels. Clinical laboratory investigations, organ weights, necropsy and histopathology results all indicate that the liver was a target organ for 1,1,3,3-tetramethyl-1,3-divinyldisiloxane. Bile duct hyperplasia, peribiliary fibrosis, and brown pigment in the bile duct were noted microscopically in the 600 mg/kg bw/day group at the end of dosing; bile duct hyperplasia was also noted for females in the 150 mg/kg bw/day group. These findings corresponded to higher mean absolute and relative liver weights and higher mean bilirubin, ALT, AST, and/or GGT levels noted in the 600 mg/kg bw/day group and were considered adverse. Under polarised light some pigment accumulations show birefringence, but this finding was not consistent in size or between animals. Pigment accumulation was considered to be an adverse finding due to the secondary bile duct hyperplasia and peribiliary fibrosis observed in the 600 mg/kg bw/day group males and females. These microscopic and organ weight changes persisted at the recovery necropsy with similar incidence and severity in the 600 mg/kg bw/day group males. In addition, hepatocellular hypertrophy was noted in the liver in all treated groups at the end of dosing; this finding was considered non-adverse. At the recovery necropsy, the incidence and severity of the hypertrophy was decreased when compared to the primary necropsy, indicating a trend toward recovery. Microscopic findings of hyaline droplets (non-adverse) were noted in the kidney for males at all dosage levels at the end of dosing. This finding progressed to hyaline droplet nephropathy (adverse) at all dosage levels. Immunohistochemistry for alpha-2u globulins was positive in the male rats with changes in distribution and morphology of the positive staining consistent with alpha-2u globulin hyaline droplet nephropathy. The hyaline droplet nephropathy persisted at the recovery necropsy. Alpha-2u globulin nephropathy is a male rat specific finding and renal effects induced in the male rats are unlikely to occur in humans. Adrenal cortical atrophy and cytoplasmic vacuolation of the pituitary were noted for males at all dosage levels at the end of doisng; these findings were considered nonadverse. The adrenal cortical atrophy corresponded to lower absolute and relative adrenal gland weights for males in the 600 mg/kg bw/day group. The cytoplasmic vacuolation observed in the pituitary at the primary necropsy was observed at the recovery necropsy with decreased incidence and severity, indicating a trend toward recovery. Adrenal cortical changes were not observed at the recovery necropsy. Based on the results of this study the NOAEL for 1,1,3,3-tetramethyl-1,3-divinyldisiloxane for systemic toxicity following oral administration in male and female rats is 50 mg/kg bw/day.

A two-week vapour inhalation study is available, which was conducted according to a protocol similar to OECD Test Guideline 412 and in compliance with GLP.The animals were exposed to 5, 50 or 246 ppm of vapour for 6 hours, 5 days a week for 10 days. There were no mortalities or treatment related toxic effect in any of the test animals. Organ weight data analysed by the Welch Trend test indicated a dose-dependent increase in female spleen weight. This increase was statistically significant in the 246 ppm exposure group when expressed as an absolute weight or as a percentage of body weight. A statistically significant increase in relative liver weight was also observed in females exposed to 246 ppm when compared with controls. No other statistically significant changes in organ weights were observed in females. The spleen and liver weight increases observed in females is of questionable toxicological significance because of the magnitude of the changes and the lack of correlation with histopathological or male data. No treatment-related changes were evident at necropsy. Histopathological examination did not reveal treatment-related effects in any tissues or organs. The changes noted in tissues of these rats were considered to be typical of incidental findings in rats of this age and strain euthanized in this manner. Since there were no treatment related deaths, the acute inhalation toxicity LD50is concluded to be higher than the highest exposure concentration of 246 ppm, equivalent to 1.9 mg/l (Dow Corning Corporation, 1993).

References:

Hard GC, Johnson KJ, Cohen SM. A comparison of rat chronic progressive nephropathy with human renal disease-implications for human risk assessment. Crit Rev Toxicol. 2009;39(4):332 346


Justification for classification or non-classification

Based on the available data 1,1,3,3-tetramethyl-1,3-divinyldisiloxane (Vi2-L2) is not classified for adverse effects following repeated exposures according to Regulation (EC) No 1272/2008.