FINAL REPORT

Version 1

Study Title

28-day repeated dose toxicity studies with 

-derivation of NOAELsAuthor

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 DISCUSSION......................................................................................... 5

REFERENCES ....................................................................................................................... 10

APPENDIX ............................................................................................................................. 11

Final Report

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, 

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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. During 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 toxicologically 

relevant. 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 considered 

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 levels. 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, no increased liver enzyme induction was observed. 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, 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 toxicity –rodent 28-dau or 14-day study”. 

Sprague-Dawley rats (CD strain) 5/sex/group received for 28 days 0, 50, 250 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. 

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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%)

HaematologyaNo toxicologically relevant findings

Blood Chemistrya

-aspartate 

aminotransferase 

(ASAT)

-alanine 

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 weight, ≥10%). Histopathological examination showed enlargement of the hepatocytes in all 

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Derivation NOAEL 28-day studies and 

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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 organ. 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 increase 

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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Derivation NOAEL 28-day studies and 

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Appendix

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 for 

read-across confirmation is lacking, no conclusion on read-across purposes for the available repeateddose data from and can be made. 

Final Report