# FINAL REPORT

Version 1

Study Title



# 28

- day repeated dose toxicity studies with

- derivation of NOAELs

Author

Completion date

19 September , 2011

Test Facility

Project Identification

- Page 1 of 12

-

### Derivation NOAEL 28-day studies and

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# Table of contents

AUTHENTICATION

.................................................................................................................. 3 1. INTRODUCTION

.............................................................................................................. 4 1.1. Preface

........................................................................................................................... 4 1.2. Scope

............................................................................................................................. 4 2. RESULTS AND DISCUSSI ON

......................................................................................... 5 REFERENCES

....................................................................................................................... 10 APPENDIX

............................................................................................................................. 11

### Derivation NOAEL 28-day studies and

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## 1. Introduction

1.1. Preface

Sponsor

Study Monitor

Test Facility



### 1.2. Scope

Two 28-day repeated dose toxicity studies are available, one study performed with the substance and one study performed with the substance For both studies NOELs were established. In this report, data from both studies were evaluated for derivation of NOAELs for both substances,

### Derivation NOAEL 28-day studies and

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## 2. Results and discussion

The study was performed according to Commission Directive 92/69/EEC (Method B7) and the Japanese Ministry of Health and Welfare Guidelines 1986 for a 28-day repeat dose oral toxicity study as required by Japanese Chemical Substances Control Law 1973 of the Ministry of International Trade and Industry (MITI) amended 1986.

Sprague-Dawley rats (Crl:CD BR strain) 5/sex/group received for 28 days 0, 15, 150 or 1000 mg/kg bw/day with Arachis oil BP as vehicle. Two recovery groups (5/sex), controls and high dose, were included for an additional 14 days observation period. Du ring the study mortality, clinical signs, body weight gain and food and water consumption were recorded. At the end of the treatment period (non-recovery group animals) or at the end of the recovery period (recovery group animals), haematology, blood chemistry and urinalysis were evaluated. Gross necropsy examination was performed on all animals, and histopathological examination of selected tissues was performed. In the table below, an overview of toxicologically relevant results is given.

0 15 150 1000 0

- recovery 1000

- recovery

m f m f m f m f m f m f Mortality none Clin signs

- fur loss together
with wetness and red/brown staining body surface

- hunched posture

- lethargy 0/5

0/5 5/5

2/5 1/5 5/5

Body weight gain No toxicologically relevant findings Water consumption No toxicologically relevant findings Food consumption No toxicologically relevant findings Haematology

- neutrophils

- platelet count i is

is Blood Chemistry

- bilirubin

- triglycerides is
ds is

Urinalysis

- volume

- spec. gravity i
d i ds d

Organ weights

- liver (%abs/%rel)

- kidneys 117/

135 101/

117 136/

136 108/

110 125/

123

Macroscopy No toxicologically relevant findings Histopathology

- hepatocyte enlargement severity

- minimal

- slight 0/5 1/5

1/5 5/5

3/5 2/5 4/5

1/5 3/5 4/5

2/5 2/5 0/5 2/5

2/5 1/5

1/5

a: blood samples from non-fasted animals i/is: increased/increased significantly d/ds: decreased/decreased significantly

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A few minutes after dosing, high dose animals showed increased salivation. This observation is considered to be related to high dose of the test substance given by gavage, and not toxicologicallyrelevant. Observed hunched posture and lethargy in high dose males are considered to be substancerelated toxicologically relevant effects

. High dose males showed a decrease in body weight gain in week 3 only. As body weight gain recovered thereafter, and overall no significant effect on body weight gain was observed, this incidental observation on body weight gain in male rats is considere d not toxicologically relevant. Urinalysis showed in high dose animals an increase in volume, which was accompanied by a decrease in specific gravity in males. In high dose animals of the recovery group, a decrease in urine volume was observed. Examination of kidney weight showed only an increase in relative kidney weight in high dose males. No histopathological changes were observed. The observed effects on relative kidney weight in males and urine volume in males and females are therefore considered not toxicologically relevant. Effects on some parameters related to liver function are observed in high dose animals, including plasma neutrophil and platelet numbers and plasma bilirubin and triglyceride le vels. In high dose animals, liver weights were increased more than 10%, both absolute and relative to body weight

. Histopathological examination of the liver showed hepatocyte enlargement in mid and high dose males and in high dose females, and his effect showed a dose-response relation in severity. Increased liver weights and hepatocyte enlargement was still observed in high-dose animals of the recovery

group

. Based on blood chemical values, n o increased liver enzyme induction was observed. Increased

liver weight of ≥10 are interpreted as potentially adverse, excep t clear association with mechanisms inducing peroxisome proliferation or mechanisms inducing a phenobarbitone-type response is involved (Andrew, 2005). As there is no evidence for mechanism of action for the observed liver effects, the effects are considered potentially adverse, and need to be considered for risk assessment. Therefore, based on the effects observed on the liver in mid and high dose animals, the oral NOAEL for sub-acute toxicity for is 15 mg/kg bw/day.

The study was designed to comply with Method B7, Annex V of the EEC Commission Directive 84/449/EEC and follows the recommendations of the OECD Guidelines for Testing of Chemicals No.407 “Repeated dose oral t oxicity

– rodent 28-dau or 14- day study”.

Sprague-Dawley rats (CD strain) 5/sex/group received for 28 days 0, 5 0, 2 50 or 1000 mg/kg bw/day

with Arachis oil BP as vehicle. During the study mortality, clinical signs, body weight gain and food and water consumption were recorded. At the end of the treatment period haematology and blood chemistry were evaluated. Gross necropsy examination was performed on all animals, and histopathological examination of selected tissues was performed. In the table below, an overview of toxicologically relevant results is given.

### Derivation NOAEL 28-day studies and

Page 7 0 50 250 1000

m f m f m f m f Mortality none Clin signs

- pilo

- erection

- hunched posture

- exophthalmos 5/5 5/5 2/5 5/5

5/5 2/5 Body weight gain d (

- 26 to

- 33%) d (

- 20 to

- 60% ) d (

- 13 to

-

14%) Water consumption i (+3 to +15%) i (+27 to +49%) i (+38 to

+119%) Food consumption d (

- 5 to

- 24%) d (

- 15 to

- 29%) Haematology a No toxicologically relevant findings Blood Chemistry a

- aspartate
aminotransferase (ASAT)

- alani ne aminotransferase (ALAT)

- glucose

- urea

- albumin

- phosphate

- potassium d is (139%)

is (145%)

is

i

is
d is

ds

is Organ weights

- liver (%abs/%rel)

- kidneys

- spleen 111/114 113/144

94/120 104/134 162/176

113/122

Macroscopy

- distended bladder 3/5 Histopathology

- hepatocyte enlargement severity

- minimal

- slight 5/5

2/5 3/5 5/5

5/5

a: blood samples from non-fasted animals i/is: increased/increased significantly d/ds: decreased/decreased significantly



### A few minutes after dosing, high dose animals showed increased salivation. This observation is considered to be related to high dose of the test substance given by gavage, and not toxicologically relevant. Observed hunched posture, pilo-erection and occasional lethargy and exophthalmos in high dose animals are considered to be substance-related toxicologically relevant effects. A decrease in body weight gain was observed in mid and high dose males and in high dose females. In males, this was accompanied by a decrease in food intake. Water consumption was increased in mid and high dose males and in high dose females. Blood chemistry investigation showed substance-related effects in high-dose animals. Plasma levels of liver enzymes ASAT and ALAT were moderately increased (139-145%) in high dose males. In addition, plasma levels of glucose, urea and albumin were increased whereas plasma phosphate levels were decreased in high dose males. In high dose female rats, increased levels of plasma urea and potassium were observed, and a decreased potassium level. Liver weights of mid and high dose females are increased, both absolute and relative to total body weight (≥10%). Also liver weight of high dose males was increased (absolute and relative to total body we ight, ≥10%). Histopathological examination showed enlargement of the hepatocytes 

## in all

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high dose animals. Increased liver weight of ≥10 are interpreted as potentially adverse, except clear association with mechanisms inducing peroxisome proliferation or mechanisms inducing a phenobarbitone-type response is involved (Andrew, 2005). As there is no evidence for mechanism of action for the observed liver effects, the effects are considered potentially adverse, and need to be considered for risk assessment. Therefore, based on the effects observed on the liver in mid and high dose animals, together with the effects on body weight gain and water and food consumption in mid dose males, the oral NOAEL for sub-acute toxicity for is 50 mg/kg bw/day.

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## 3. Conclusion

Two 28-day repeated dose toxicity studies are available, one study performed with the substance

and one study performed with the substance In both studies, comparable toxicological effects were observed, with liver as target orga n. In the absence of clear evidence for a

non -adverse mode of action, the observed effect on the liver are considered potentially adverse.

For the NOAEL for subacute oral toxicity is 15 mg/kg bw/day, based on hepatocyte enlargement in males dosed 150 mg/kg bw/day and 1000 mg/kg bw/day, with a dose-related in crease in severity. In the 1000 mg/kg bw/day group in which female rats also showed hepatocyte enlargement a significant increase in liver weight was observed.

For the NOAEL for subacute oral toxicity is 50 mg/kg bw/day, based on a dose-related increased liver weight in females dosed 250 mg/kg bw/day and 1000 mg/kg bw/day, and increased liver weight in males dosed 1000 mg/kg bw/day. In high dose animals, histopathological examination of the liver showed hepatocyte enlargement.

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## References

Andrews D (2005). PSD Guidance Document: Interpretation of liver enlargement in regulatory toxicity

studies.

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## Append ix

Read-across

General approach

In the REACH Regulation (EC) No 1907/2006, Annex XI deals with the general rules for adaptation of the standard testing regime. According to Annex XI 1.5, it is possible to demonstrate that testing does not appear scientifically necessary based on grouping of substances and read-across approach. Substances whose physic-chemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group or “category” of substances. The similarities may be based on:

- A common functional group

- The common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals; or

- A constant pattern in changing of the potency of the properties across the category.

This means that for read-across evaluation, similar structures, same functional groups and similar available physico-chemical and (eco)toxicological properties are indispensable.

This is also outlined in the ECHA guidance on information requirements and chemical safety assessment, Chapter R6 (see attached guidance in the e-mail section 6.2.2.1 for more details).

Human toxicological endpoints

In order to use a read-across approach for human toxicological endpoints, it needs to be demonstrated that the substances to be concerned for read-across approach behave in a comparable way after exposure. As generally reliable toxicokinetic data (data on adsorption, distribution, metabolism, excretion) are lacking, reliable information on the physic-chemical properties that are relevant for biological effects needs to be provided in order to allow making robust assumptions on the fate of the substance in biological systems.

This means that reliable information on physico-chemical properties of all read-across substances is essential to make an assumption on comparable toxicokinetic behaviour of the substances (comparable water solubility, partition coefficient octanol/water, vapour pressure (liquid/gas), particle size (solid). To this end, QSAR predictions are not recommended, as data on these endpoints are crucial for the read-across substantiating. Therefore, reliable physico-chemical testdata needs to be available for a read-across approach.

In addition to demonstrate comparable physic-chemical properties and toxicokinetic behavior, also comparable toxicodynamic behavior needs to be assumed. This will only be reliable for systemic effects. In needs to be demonstrated that after exposure, the toxicological effect is comparable, meaning that exposure will result in observation of the most relevant systemic effect in the same target organ (at the same order of exposure level).

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As outlined above, read-across assumption for human toxicological endpoints requires reliable data for both physic-chemical endpoints as well as for toxicological endpoints. For the available set of reliable data is limited, and is not valid for read-across evaluation yet. As essential information forread-across confirmation is lacking, no conclusion on read-across purposes for the available repeateddose data from and can be made.