Dibutyl itaconate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 November 2021 - 03 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

25 June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl itaconate

EC Number:

218-451-9

EC Name:

Dibutyl itaconate

Cas Number:

2155-60-4

Molecular formula:

C13H22O4

IUPAC Name:

1,4-dibutyl 2-methylidenebutanedioate

Specific details on test material used for the study:

Physical description: Clear yellow liquid
Storage conditions: At room temperature
Test item handling: No specific handling conditions required
Volatile: Yes, vapour pressure: 0.19 Pa @ 25°C
pH: 7
Specific gravity/density: 0.985 g/mL

Test animals

Species:

rat

Strain:

other: Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 130 – 173 g males; 104 – 137 g females (targets: 100 to 300 g males and 100 to 200 g females)
- Fasting period before study: Not reported
- Housing: Polycarbonate cages containing sterilized wooden fibers as bedding material equipped with water bottles. Up to 5 animals of the same sex and same dosing group together. Animals will be socially housed for psychological/environmental enrichment and may be provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet: ad libitum, SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
- Water: ad libitum, municipal water
- Contamination: It is considered that there are no known contaminants in the feed or water that would interfere with the objectives of the study.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 to 22°C (actual range); 18 to 24°C (target range)
- Humidity: 38 to 56% (actual range); 40 to 70% (target range); The values that were outside the targeted mean humidity range occurred for 1 day and was without a noticeable effect on the clinical condition of the animals or on the outcome of the study.
- Air changes: Ten or more air changes per hour
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 29 November 2021 To: 1 March 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

specific gravity 1.125

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dose formulations were divided into aliquots where required to allow to be dispensed on each dosing occasion. An adjustment was made for specific gravity of the test item and vehicle. No correction was made for the purity/composition of the test item or vehicle.
The dosing formulations were prepared at least weekly, filled out in daily portions and stored in the refrigerator protected from light. Before dosing they were removed from the refrigerator and stirred at room temperature for at least 30 min.

VEHICLE
- Concentration in vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Remarks:

See table 1 under 'Any other information on materials and methods incl. tables'

Details on analytical verification of doses or concentrations:

Analyses was performed by using a validated analytical procedure (Test Facility Study No. 20299095).

Acceptance criteria for concentration: mean sample concentration results within or equal to ± 10% of theoretical concentration.
Acceptance criteria for homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group.

Stability analyses performed previously in conjunction with the method development and validation (Test Facility Study No. 20299095) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on results of a 14-day repeated dose toxicity study with oral exposure of the test material in rats (Test Facility Study No. 20299096).
- Fasting period before blood sampling for clinical biochemistry: Yes (overnight with a maximum of 24 hours)

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule mortality check: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency will be at least once daily.
- Time schedule observations: At least once daily; from Day 1 at 0 to 1 hours postdose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pretreatment and weekly; from Week 1 and throughout the study, and on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study. In order to monitor the health status, animals may be weighed more often.

FOOD CONSUMPTION: Yes
- Time schedule: Weekly; from at least Day 1 and throughout the study

WATER CONSUMPTION: Yes
- Time schedule: Regular basis throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment Period - All Main Study and (including spare animals)
Dosing Period - All Group 1 and 4 Main Study animals during Week 13. If treatment-related findings are noted, the other animals will also be examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Sampled between 7.00 and 10.30 from the retro-orbital sinus at the end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: all main animals
- Parameters in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Sampled between 7.00 and 10.30 from the retro-orbital sinus at the end of treatment
- Animals fasted: Yes (overnight)
- How many animals: All main animals
- Parameters in table 3 were examined.

SERUM HORMONES: Yes
- Time of blood sample collection: Sampled between 7.00 and 10.30 from the retro-orbital sinus at the end of treatment
- Animals fasted: Yes (overnight)
- How many animals: All main animals

COAGULATION: Yes
- Time schedule for collection of blood: Sampled between 7.00 and 10.30 from the retro-orbital sinus at the end of treatment
- Animals fasted: Yes (overnight)
- How many animals: All main animals
- Parameters in table 4 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the Dosing Period.
- Dose groups that were examined: All dose groups, the first 5 animals per sex per group during Week 13.
- Battery of functions tested: hearing ability, pupillary reflex and static righting reflex, fore- and hind-limb grip strength, locomotor activity

IMMUNOLOGY: No

ESTROUS STAGE DETERMINATION: Yes
- Time schedule: end of treatment on day of necropsy, a vaginal smear was taken to determine the stage of estrus from all main study animals

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, on all animals

HISTOPATHOLOGY: Yes, for animals found dear or unscheduled euthanasia, group 1 and group 4 the full histopathology was performed (see table 5). For group 2 and 3 only gross lesions and specified target tissues were evaluated.

ORGAN WEIGHTS: Yes (see table 5)

Statistics:

Inferential Statistical Methods
All statistical tests will be conducted at the 5% significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric/non-parametric:
Levene’s test will be used to assess the homogeneity of group variances.
The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s or Dunn’s test, respectively.

Non-parametric:
The groups will be compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons will be conducted using Dunn’s test.

ANCNOVA:
The data corresponding to a response variable of interest and to a related covariate will be submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons will be conducted using Dunnett’s test.

Incidence:
A Fisher’s exact test will be used to conduct pairwise group comparisons of interest.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related clinical signs were noted during clinical observations or during weekly arena observations.
Salivation was seen after dosing among most animals dosed at 100, 300 and 1000 mg/kg bw/day during the Treatment Period and in a few control animals. Taking into account the nature and minor severity of the effect and its time of occurrence (i.e., after dosing), this sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the Treatment Period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and/or did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test material.
One female at 100 mg/kg bw/day was found dead on Day 55. Moderate salivation was observed after dosing on the day of death. Macroscopic findings included an abnormal clear watery content in the thoracic body cavity and perforation of the esophagus. This indicates that the cause of death was perforation of the esophagus seen macroscopically. It was not observed microscopically. Collectively, these findings were consistent with a gavage-related death and the cause of death was considered not related to treatment with the test material.
One female at 100 mg/kg bw/day died during anesthesia for blood sampling on Day 93. Since there were no macroscopic or microscopic findings for this animal, this dead was considered accidental and not related to treatment with the test material.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment with the test material.
Any statistically significant changes in body weight gain (e.g., in males at 300 and 1000 mg/kg bw/day in the first week) were considered to be unrelated to the test material since no trend was apparent regarding dose and/or duration of dosing.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test material-related changes in food consumption were recorded.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to the test material.
The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to the test material.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology parameters were considered unaffected by treatment with the test material in males up to 1000 mg/kg bw/day and in females up to 300 mg/kg bw/day.
In females, lower platelet counts (0.84x of control) and mean corpuscular volumes (0.97x,) were noted at 1000 mg/kg bw/day. Based on the absence of a histopathological correlation, these changes in haematology parameters were considered to be non-adverse.
Remaining differences in hematology parameters, regardless of statistical significance, were considered not test material-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Coagulation parameters were considered to be unaffected by treatment with the test material. Any statistical changes in coagulation parameters were considered not test material-related on the absence of a dose response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The changes are presented in table 6 under 'Any other information on results incl. tables'.
At 1000 mg/kg bw/day, alkaline phosphatase activity was increased in males and females, see Table 6 Test item-Related Clinical Chemistry Changes for the ratio difference. In addition, total bilirubin concentration was increased in males and females. Furthermore, creatinine concentration was decreased in males at 100, 300 and 1000 mg/kg bw/day and in females at 300 and 1000 mg/kg bw/day. Glucose concentration was decreased in males at 1000 mg/kg bw/day and in females at 300 and 1000 mg/kg bw/day.
Cholesterol concentration was decreased in males at 300 and 1000 mg/kg bw/day and in females at 1000 mg/kg bw/day. Accordingly, HDL and LDL cholesterol concentrations were also decreased in males at 300 and 1000 mg/kg bw/day and in females at 1000 mg/kg bw/day.
Potassium concentration was increased in males at 100, 300 and 1000 mg/kg bw/day. In addition, chloride concentration was decreased in males at 1000 mg/kg bw/day and in females at 100, 300 and 1000 mg/kg bw/day. Inorganic Phosphate concentration was increased in males at 300 and 1000 mg/kg bw/day. These clinical chemistry parameters were considered to be non-adverse based on the absence of any histopathological correlation.
Remaining differences in clinical chemistry parameters, regardless of statistical significance, were considered not test material-related based on the absence of a dose response, general overlap of individual values with the range of control values, a lack of biological relevance and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones were considered to be unaffected by treatment with the test material in animals up to 300 mg/kg bw/day.
At 1000 mg/kg bw/day, thyroxine (T4) was decreased in males (0.78x of control) and triiodothyronine (T3) was decreased in females (0.69x). T4 mean values in males at 1000 mg/kg bw/day were outside the historical control range, while T3 mean values in females remained within the historical control range. Under the conditions of this study no adverse effect was observed that could be linked to the reduction of T4 and therefore this reduction was not taken into account when determining the NOAEL. As mean values of T3 remained within the normal range of variation, this was considered non-adverse.
Thyroid-stimulating hormone (TSH) was considered to be unaffected by the treatment with the test material. The apparent increase in TSH in females at all dose levels was mainly due to a low control value. The means were not outside the historical control range and therefore not test material-related.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were test material-related alterations in organ weights of the thyroid gland, liver and kidney as shown in table 7 Mean Percent Organ Weight Differences from Control Group.
Test material-related higher thyroid gland weights (absolute and relative to body weights) were noted in females at 1000 mg/kg bw/day. This correlated microscopically with follicular cell hypertrophy.
Test material-related higher liver weights (absolute and relative to body weights) were noted in males at 300 and 1000 mg/kg bw/day and in females at 1000 mg/kg bw/day. This correlated macroscopically with enlarged liver and microscopically with centrilobular hepatocellular hypertrophy and periportal hepatocellular vacuolation.
Test material-related higher kidney weights (absolute and relative to body weights) were noted in males at 300 and 1000 mg/kg bw/day and in females at 100, 300 and 1000 mg/kg bw/day. This correlated macroscopically with enlarged kidney and microscopically with tubular vacuolation.
Lower pituitary gland weights (absolute and relative to body weights) were noted females at 1000 mg/kg bw/day. This lacked a macroscopic or microscopic correlate. The relationship with the test material cannot be excluded, however, in absence of a macroscopic or microscopic correlate, this finding was considered to be non-adverse.
Based on the histopathological findings, the increased liver and kidney weights and the decreased pituitary gland weights are expected to recover once treatment with the test item has ended. However, since there was no recovery group included in this study there is no data to support this hypothesis.

Any other differences, including those that reached statistical significance (heart in males at 1000 mg/kg bw/day and females at 300 mg/kg bw/day) 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:

effects observed, treatment-related

Description (incidence and severity):

There were test material-related gross observations in the liver and kidney.
Enlargement of the liver was noted in 1/10 males at 1000 mg/kg bw/day, which correlated with increased liver weight and microscopically with centrilobular hepatocellular hypertrophy. Pale discoloration of the liver was noted in 1/10 males at 1000 mg/kg bw/day and in 1/10 females at 300 mg/kg bw/day and in 7/10 females at 1000 mg/kg bw/day. Pale discoloration generally correlated with increased liver weight and with periportal hepatocellular vacuolation seen microscopically in the liver.
Enlargement of the kidney was noted in 7/10 males at 1000 mg/kg bw/day, which generally correlated with increased kidney weight and with tubular vacuolation seen microscopically.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between test material-treated and control animals, and all animals showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
In males, decreased hindleg grip strength at 300 and 1000 mg/kg bw/day and decreased foreleg grip strength at 1000 mg/kg bw/day (not statistically significant) were noted. These findings were considered not to be test material-related based on that the values remained in the range expected for these animals of this strange and age.
In females, decreased mean movements and mean ambulations at 1000 mg/kg bw/day (not statistically significant) were noted. These findings were considered not to be test material-related based on that the values remained in the range expected for these animals of this strange and age.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were test material-related microscopic observations in the thyroid gland, liver and kidney and they are summarized in table 8 Summary Test Material-Related Microscopic Findings.
In the thyroid gland, an increased incidence of minimal follicular cell hypertrophy was recorded in males and females at 1000 mg/kg bw/day. Hypertrophy generally correlated with increased thyroid weight in females. Since hepatic enzymes play a role in the metabolism of the thyroid, there is a relationship between the liver findings (higher liver weight, enlarged liver, centrilobular hepatocellular hypertrophy and increased liver parameters) and the thyroid finding (higher thyroid weight, follicular cell hypertrophy and increased thyroid hormone) (Greaves, 2012). Findings in the thyroid gland were regarded secondary to the liver findings.
In the liver, minimal to moderate periportal hepatocellular vacuolation was noted in males starting at 300 mg/kg bw/day and females starting at 100 mg/kg bw/day. Vacuolation generally correlated with increased liver weight and pale discoloration seen macroscopically. Minimal centrilobular hepatocellular hypertrophy was noted in males at 1000 mg/kg bw/day. Hypertrophy generally correlated with enlarged liver and increased liver weight.
In the kidney, minimal to mild tubular vacuolation was noted in males at 1000 mg/kg bw/day and females starting at 300 mg/kg bw/day. Vacuolation generally correlated with enlarged kidney and increased kidney weight.
All histopathological findings (follicular cell hypertrophy in the thyroid gland, periportal hepatocellular vacuolation and centrilobular hepatocellular hypertrophy in the liver, and tubular vacuolation in the kidney) were considered to be non-adverse based on their (non-degenerating) nature and/or degree of severity (minimal hypertrophy in the thyroid and liver, minimal to mild or moderate vacuolation in the liver and kidney).

There were no other test material-related histologic changes. 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 material related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse test material-related alterations observed.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

DOSE FORMULATION ANALYSIS

Accuracy

The test item concentrations analyzed in the formulations of Groups 2-4 prepared for use in Weeks 1 and 12 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90-110% of target concentration).

For the formulation of Groups 2 and 4 prepared for use in Week 6, the mean concentrations were 87% and 88% of target. An Out of Specification investigation was performed to determine the cause of the relatively low recoveries. Given the fact that the mean accuracies of the QC samples were comparable to the mean accuracies of the respective samples, the results were accepted. As the results between the QC samples and formulation samples were comparable it suggests that the observed low recoveries were likely due to analytical reasons. Therefore, it was considered that the formulations were accurately prepared throughout the study.

No test material was detected in the Group 1 formulations.

Homogeneity

The formulations of Group 2 and Group 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

 

TABLES

Table 6. Test item-Related Clinical Chemistry Changes

Groups	2		3		4
Dose (mg/kg bw/day)	100	100	300	300	1000	1000
Sex	M	F	M	F	M	F
Alkaline phosphatase
End of Treatment	–	–	–	–	2.24**	2.34**
Bilirubin
End of Treatment	–	–	–	–	1.53	1.31**
Creatinine
End of Treatment	0.87**	–	0.86**	0.80**	0.81**	0.82**
Glucose
End of Treatment	–	–	–	0.77**	0.73**	0.79**
Cholesterol
End of Treatment	–	–	0.68**	–	0.48**	0.47**
HDL cholesterol
End of Treatment	–	–	0.75**	–	0.53**	0.44**
LDL cholesterol
End of Treatment	–	–	0.71**	–	0.47**	0.27**
Potassium
End of Treatment	1.11**	–	1.11**	–	1.15**	–
Chloride
End of Treatment	–	0.98*	–	0.97**	0.97**	0.96**
Inorganic Phosphate
End of Treatment	–	–	1.18**	–	1.31**	–

M = Males F = Females
A dash (—) indicates absence of change. Numerical values indicate fold change of the treated group mean value relative to the control group mean value. *= P ≤ 0.05; **=P ≥ 0.01.

 

Table 7. Mean Percent Organ Weight Differences from Control Group

	Males					Females
Dose level (mg/kg bw/day):	100		300		1000	100	300	1000
THYROID GLAND
Absolute	11		4		17	8	14	26*
Relative to body weight	12		3		23	1	11	26**
LIVER
Absolute	3		18**		28**	9	10	36**
Relative to body weight	4		16**		33**	2	7	37**
KIDNEY
Absolute	12		23**		45**	19*	17*	40**
Relative to body weight	12		21**		51**	11**	14**	40**
PITUITARY GLAND
Absolute	-13		-2		-15	-2	-7	-17*
Relative to body weight	-12		-3		-11	-8	-8	-18**

*: P≤0.05, **: P≤0.01

 

Table 8. Summary Test Material-Related Microscopic Findings

	Males				Females
Dose level (mg/kg bw/day):	0	100	300	1000	0	100	300	1000
THYROID GLAND ^a	10	10	10	10	10	9	10	10
Hypertrophy; follicular cell
minimal	1	1	1	4	1	-	1	4
LIVER ^a	10	10	10	10	10	9	10	10
Vacuolation; hepatocellular,      periportal
minimal	-	-	5	5	-	1	6	1
mild	-	-	-	3	-	-	1	7
moderate	-	-	-	2	-	-	-	1
Hypertrophy; centrilobular,     hepatocellular
minimal	-	-	-	6	-	-	-	0
KIDNEY ^a	10	10	10	10	10	9	10	10
Vacuolation; tubular
minimal	-	-	-	5	-	-	6	4
mild	-	-	-	-	-	-	1	6

^a  =  Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:

A repeated dose toxicity study was performed in rats according to OECD TG 408 and in accordance with GLP principles. In conclusion, administration of the test material by once daily oral gavage was well tolerated in Wistar Han rats up to 1000 mg/kg bw/day. There were no adverse test material related alterations observed.
Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be at least 1000 mg/kg bw/day in males and females.
In this study, a marked reduction of T4 was observed in males at 1000 mg/kg bw/day. However, under the conditions of this study no adverse effect was observed that could be linked to the reduction of T4 and therefore this reduction was not taken into account when determining the NOAEL.

Executive summary:

A repeated dose toxicity study was performed in rats according to OECD TG 408 and in accordance with GLP principles. 

The objective of this study was to determine the potential toxicity of the test material, when given orally by gavage for 90 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

The dose levels were selected based on results of a 14-day repeated dose toxicity study with oral exposure of the test material in rats (Test Facility Study No. 20299096). The study design was as follows:

Group No.	Dose Level (mg/kg bw/day)	Dose Volume ^a (mL/kg)	Number of Animals
			Males	Females
1	0 (Vehicle)	5	10	10
2	100	5	10	10
3	300	5	10	10
4	1000	5	10	10

^a Based on the most recent body weight measurement.

Chemical analyses of formulations were conducted in Weeks 1, 6 and 12 to assess accuracy and homogeneity. Formulation analyses confirmed that formulations were prepared accurately and homogenously for all occasions.

The following parameters and end points were evaluated in this study: mortality, clinical signs, functional observations, body weights, food consumption, ophthalmology, estrous stage determination, clinical pathology parameters (haematology, coagulation, clinical chemistry), macroscopic examination, organ weights and microscopic examination.

No test material-related mortality occurred during the study.

One female at 100 mg/kg bw/day was found dead on Day 54 and one female at 100 mg/kg bw/day died during anesthesia on the day of scheduled necropsy. Both deaths were considered to be accidental and not related to treatment with the test material.

At 100 mg/kg bw/day, non-adverse clinical pathology changes consisted of increased potassium and decreased creatinine concentrations in males and decreased chloride concentration in females. Non-adverse histopathological changes consisted of minimal periportal hepatocellular vacuolation in the liver of females. In addition, a non-adverse higher kidney weight was seen in females.

At 300 mg/kg bw/day, non-adverse clinical pathology changes consisted of increased potassium and inorganic phosphate concentration. In addition, a non-adverse decreased creatinine, glucose and chloride was noted in females and a decreased cholesterol HDL and LDL cholesterol concentration in males. Non-adverse histopathological changes consisted of minimal to mild periportal hepatocellular vacuolation in the liver of males and females with correlating higher liver weight (males) and macroscopic pale discoloration (females). In addition, a non-adverse minimal to mild tubular vacuolation was observed in females with correlating higher kidney weights (also higher in males).

At 1000 mg/kg bw/day, non-adverse clinical pathology changes consisted of increased alkaline phosphatase activities and total bilirubin concentrations in males and females and decreased creatinine, glucose, chloride, cholesterol, HDL and LDL cholesterol in males and females. In addition, potassium and inorganic phosphate are non-adverse increased in males. In addition, non-adverse decreases in platelet counts and mean corpuscular volumes were noted in females. Hormone analysis showed decreased T4 levels in males with mean values below the normal range of variation. Non-adverse decreased T3 levels were noted in females at 1000 mg/kg bw/day.
Non-adverse histopathological changes were noted in the liver and consisted of minimal to moderate periportal hepatocellular vacuolation in males and females and minimal centrilobular hepatocellular hypertrophy in males with correlating higher liver weight and macroscopic pale discoloration or macroscopic enlargement. Furthermore, non-adverse minimal to mild tubular vacuolation was observed in the kidney of males and females with correlating macroscopic enlarged kidney (males) and higher kidney weight (males and females). In addition, minimal follicular cell hypertrophy in the thyroid gland was observed in males and females with correlating higher thyroid gland weight in females. Lastly, lower pituitary gland weights without a macroscopic or microscopic correlate were noted in females.

No test material-related changes were noted in any of the remaining parameters investigated in this study (i.e., clinical observations, body weight, food consumption, functional observations, ophthalmoscopy and coagulation).

In conclusion, administration of the test material by once daily oral gavage was well tolerated in Wistar Han rats up to 1000 mg/kg bw/day. There were no adverse test material‑related alterations observed.

Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be at least 1000 mg/kg bw/day in males and females.

In this study, a marked reduction of T4 was observed in males at 1000 mg/kg bw/day. However, under the conditions of this study no adverse effect was observed that could be linked to the reduction of T4 and therefore this reduction was not taken into account when determining the NOAEL.