Registration Dossier

Administrative data

Description of key information

In a sub-chronic oral toxicity study (OECD 408), three groups of 10/sex Han Wistar rats received the substance at doses of 100, 300 and 1000 mg/kg/day. A similarly constituted control group received the vehicle (1% w/v methylcellulose) at the same volume-dose as the treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by daily visual observation), ophthalmic examination, hematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

Results

The appearance and behavior of the animals, sensory reactivity and motor activity were unaffected by treatment and there were no treatment-related deaths.

At the grip strength investigations in Week 12, there was a trend towards low hind limb grip strength in the males, which was statistically significant at 1000 mg/kg/day.

Body weight gain and food consumption were significantly low in males receiving 1000 mg/kg/day and body weight gain was low in females receiving 1000 mg/kg/day.

There were no treatment-related ophthalmic findings.

At the hematological examination performed in Week 13, there were some reductions of leucocyte parameters which attained statistical significance, particularly in the males receiving 1000 mg/kg/day (low lymphocyte, eosinophil, basophil, monocyte, neutrophil and large unstained cell counts with a consequential decrease of total leucocyte count); there was a similar, but less pronounced effect in the females receiving 1000 mg/kg/day, where lymphocyte, eosinophil and neutrophil counts were low, resulting in a consequential decrease of total leucocyte count. 

At the biochemical examination of the blood plasma, plasma alkaline phosphatase activity was high in males and females receiving 1000 mg/kg/day and bile acid concentrations were high in males receiving 300 or 1000 mg/kg/day and in females receiving 1000 mg/kg/day. Plasma cholesterol concentrationswere low in males and females receiving 1000 mg/kg/day and plasma creatinine concentration was high in males receiving 1000 mg/kg/day.

After 13 weeks of treatment, liver and kidney weights were high in both sexes given 1000 mg/kg/day and epididymis weights were slightly low in males given 1000 mg/kg/day. Macroscopic findings after 13 weeks of treatment were confined to the presence of depressions on the non‑glandular stomach in one male and one female given 1000 mg/kg/day. 

Treatment-related histopathological changes occurred in the stomach, liver and thyroid gland. In the stomach, hyperplasia of the non-glandular epithelium was seen in males and females given 1000 mg/kg/day and ulceration of the non-glandular epithelium was seen in one high dose male. In the liver, centrilobular hypertrophy was recorded in males and females given 1000 mg/kg/day and in the thyroid gland, follicular cell hypertrophy was seen in two males given 1000 mg/kg/day.

Conclusion

It is concluded that the substance was well-tolerated, but at 1000 mg/kg/day caused local irritation to the gastric epithelium and hepatocytic hypertrophy that was likely due to adaptive enzyme induction and secondary to rodent-specific hypertrophic changes in the thyroid. Based on these effects and effects on body weight gain, white blood cells and blood biochemistry, the no-observed-adverse-effect-level (NOAEL) is considered to be 300 mg/kg/day.

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:
26 April 2017 to 29 March 2018
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)
Version / remarks:
revised 1998
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:CD(SD) rat
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: RccHan™;WIST rat
- Source: Envigo RMS Limited
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 41-47 days
- Weight at study initiation: males: 128-170 g; females: 112-145 g
- Housing: 5/sex/cage in polycarbonate cages with a stainless steel mesh lid (wood based bedding and Aspen chew block/Plastic shelter)
- Diet: Teklad 2014C pelleted diet ad libitum
- Water: tap water ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY: certificates of analysis of diet and water available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air changes (per hr): no data (Filtered fresh air which was passed to atmosphere and not recirculated)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 4 September 2017: To: 5 December 2017
Route of administration:
oral: gavage
Details on route of administration:
using a suitably graduated rubber-free syringe and a plastic catheter inserted via the mouth.
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: the substance was weighed and then mixed with the vehicle (approximately 50% of the final volume). The mixture was magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous

VEHICLE:1% CMC
- Concentration in vehicle: 0, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For Week 1 and Week 12, freshly prepared test formulations were sampled (4 × 1 mL,accurately weighed) and submitted for analysis. Duplicate samples were analyzed. The samples were dissolved using ultrasonic vibration and swirling in a suitable volume of diluent (Methanol / water 80/20 v/v). The extract was diluted using diluent (where necessary) to provide a solution containing the substance at an expected concentration within the range 2 µg/mL to 4 µg/mL.

Method:
High performance liquidchromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector.
Column: Agilent Poroshell C18-EC, 2.7 µm, 100 × 4.6 mm
Column temperature: 45ºC
Sample temperature: Ambient.
Mobile Phase: Methanol / water 85 / 15 v/v
Flow rate: 1 mL/min
Needle wash: Methanol / water 80 / 20 v/v
Detector wavelength: UV, 229 nm
Injection volume: 10 µL
Run time: 6 minutes
Approximate retention time: 4.6 minutes

Results method validation:
Calibration linearity (range 1-5 ug/mL): r > 0.999
Specificity: absence of peak for substance in control sample
Precisions calibration (CV 0.22% 1 ug/mL, 0.28% 5 ug/mL)
Accuracy: procedural recovery value of 101.6% (CV=1.13%, n=5) was obtained for 1 mg/mL and 101.0% (CV=0.13%, n=5) was obtained for 200 mg/mL.
Stability (15 days 4 ºC): 1 mg/mL 96% of initial; 200 mg/mL 97% of initial
Stability (1 day at 21 ºC): 1 mg/mL 105% of initial; 200 mg/mL 103.5% of initial
Homogeneity: CV < 5.6% at 1 mg/mL; CV < 1% at 200 mg/mL
LOQ: 2.2 ng/mL and 7.4 ng/mL

Preparation analyses: see below
Procedural recovery: 98.1-104.5% at 1 and 200 mg/L
Accuracy: week 1: 102-109% of nominal for all concentrations; week 12: 91.3-94.7% of nominal for all concentrations
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
measured 94.7-102% of nominal
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
measured 91.3-106.3% of nominal
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
measured 94.2-109% of nominal
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 2-week dose range finding study
Envigo Study No. QH95RV. In that study, dose levels of 500, 750 and 1000 mg/kg/day were well tolerated, with no premature deaths and no effect of treatment upon the general health, behavior or appearance of the animals. The overall weight gain of males and females receiving 1000 mg/kg/day was slightly low, but food consumption was unaffected and the biochemical examination of plasma revealed only slightly low alanine amino-transferase activity and cholesterol concentration in males and low urea concentration in females, none of which were considered to be of toxicological importance. At the macroscopic examination, stomach depressions were identified in one male and two females given 750 mg/kg/day and liver weights were high in males given 750 or 1000 mg/kg/day and in females at the high dose.
Based on this information, it was considered appropriate to investigate a high dose level of 1000 mg/kg/day (equivalent to the limit dose for this study type) on the current study. The intermediate and low dose levels of 300 and 100 mg/kg/day were selected to assess the dose responsiveness of any test item-related findings following a longer (i.e. 13 week) duration of treatment.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DOSING OBSERVATIONS: Pre-dose and 1-2 hours after completion of dosing of all groups (during week 1 also at the end of the working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: in a standard arena before treatment commenced and during each week of treatment, detailed physical/behaviour examinations

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 and weekly thereafter

FOOD CONSUMPTION : Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes (visual inspection daily)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment (all animals) and week 12 (controls and high dose animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes overnight
- How many animals: all
- Parameters checked: Hematocrit (Hct), Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count:, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (PT),Activated partial thromboplastin time (APTT)
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes overnight
- How many animals: all
- Parameters checked: Alkaline phosphatase (ALP),Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Bile acids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Total protein (Total Prot), Albumin (Alb)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 12
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (includes: approach response, pinna reflex, auditory startle reflex, tail pinch)/ grip strength (fore- and hindlimb) / motor activity(beam crossing over 6 min intervals for 1 hour)

IMMUNOLOGY: No

Sacrifice and pathology:
ORGAN WEIGHTS: Yes (all animals, see table)

GROSS PATHOLOGY: Yes (all animals see table)

HISTOPATHOLOGY: Yes (see table)
Full List All animals killed or dying prematurely and all animals of Groups 1 and 4 killed at the end of the treatment period.
Liver and stomach All animals of Groups 2 and 3 killed at the end of the treatment period.
Thyroid gland All males of Groups 2 and 3 killed at the end of the treatment period.


Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following sequence of statistical tests was used for grip strength, motor activity, body weight, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.
For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means to correct any influence of body weight.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
no effects observed
Description (incidence and severity):
The appearance and behaviour of the animals was unaffected by treatment and no signs were recorded in association with dose administration.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1 male at 300 mg/kg bw on day 18 (gavage error)
1 male at 1000 mg/kg bw on day 12 (gavage error)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall body weight gain (Week 1 to 13) was statistically significantly low, when compared with controls, in males and females receiving 1000 mg/kg/day (87 and 88% of control for males and females, respectively).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was slightly low, when compared with control, in males receiving 1000 mg/kg/day (91% of control), but statistical significance was not attained and there was no similar effect in the females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
no abnormalities reported
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related ophthalmic findings.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males 1000 mg/kg bw: statistically significantly low lymphocyte, eosinophil, basophil, neutophil, monocyte and large unstained cell counts in males receiving 1000 mg/kg/day (69, 43, 50, 73, 79 and 33% of control, respectively).Total leucocyte count sign decreased (69% of control)
Males 300 mg/kg bw: decreased neutophil count (73% of control)
Females 1000 mg/kg bw: decreased lymphocyte and eosinophil count (94% and 63% of control, respectively). Total leucocyte count decreased (78% of control)
Females 300 mg/kg bw: decreased lymphocyte count (79% of control). Total leucocyte count 95% of control

All inter-group differences on erythrocyte count, hematocrit and hemoglobinfrom control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. The statistically significant decrease of activated partial thromboplastin time in females receiving 1000 mg/kg/day which was principally attributed to one female.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
males 1000 mg/kg bw: sign increase ALP (36%), bile acids (65%), creatinine (15%); sign decrease cholesterol (54%)
males 300 mg/kg bw: sign increase bile acids (62%)
males 100 mg/kg bw: increase bile acids (9%)
Females 1000 mg/kg bw: sign increase ALP (27%), bile acids (95%); sign decrease cholesterol (23%)
Females 300 mg/kg bw: increase bile acids (29%); decrease cholesterol (14%)
Females 100 mg/kg bw: increase bile acids (45%)

All other inter-group differences from control, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity observations: no treatment related effects

Mean hind limb grip strength: sign decreased at 1000 mg/kg bw in males. The individual mean values were, however, generally within the background range (0.37 to 0.63 kg; 21 studies) with the exception of one male receiving 300 mg/kg/day and one male receiving 1000 mg/kg/day
Forelimb grip strength: non-sign decreased in males at 1000 mg/kg/day (no clear dose-relationship). All individual mean values were within the background range (0.70 to 1.28 kg; 21 studies).

Motor activity: no treatment related effects
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
males: 1000 mg/kg bw sign increased relative kidney and liver weight (17 and 32%); sign decreased epididymides weight (8%)
females: 1000 mg/kg bw sign increased relative kidney and liver weight (7 and 35%)

All other differences from controls, including those attaining statistical significance, were minor and were attributed to normal biological variation. Such differences included the slightly low brain weights in males given 1000 mg/kg/day and slightly low thymus weights in females given 100 or 1000 mg/kg/day, where there was no dose relationship.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Depressions were seen on the non-glandular region of the stomach in one male and one female given 1000 mg/kg/day.
Summary of findings in the stomach for animals killed after 13 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 300 1000 0 100 300 1000
Depression(s) 0 0 0 1 0 0 0 1
Number of tissues examined 10 10 9 9 10 10 10 10

The incidence and distribution of all other findings were considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males 1000 mg/kg bw : hyperplasia of the non-glandular epithelium of the stomach (5/9) minimal ulceration of the non-glandular epithelium of the stomach (1/9); Centrilobular hypertrophy in the liver (4/9); Minimal follicular cell hypertrophy of the thyroid (2/9)
Females 1000 mg/kg bw: hyperplasia of the non-glandular epithelium of the stomach (4/10); Centrilobular hypertrophy in the liver (3/10)
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
no treatment related effect (oestrus cycle 4-5 days pre-treatment, at termination all females were in diestrous phase)
Details on results:
The appearance and behavior of the animals, sensory reactivity and motor activity were unaffected by treatment and there were no treatment-related deaths.
At the grip strength investigations in Week 12, there was a trend towards low hind limb grip strength in the males, which was statistically significant at 1000 mg/kg/day.
Body weight gain and food consumption were significantly low in males receiving 1000 mg/kg/day and body weight gain was low in females receiving 1000 mg/kg/day.
There were no treatment-related ophthalmic findings.
At the hematological examination performed in Week 13, there were some reductions of leucocyte parameters which attained statistical significance, particularly in the males receiving 1000 mg/kg/day (low lymphocyte, eosinophil, basophil, monocyte, neutrophil and large unstained cell counts with a consequential decrease of total leucocyte count); there was a similar, but less pronounced effect in the females receiving 1000 mg/kg/day, where lymphocyte, eosinophil and neutrophil counts were low, resulting in a consequential decrease of total leucocyte count. 
At the biochemical examination of the blood plasma, plasma alkaline phosphatase activity was high in males and females receiving 1000 mg/kg/day and bile acid concentrations were high in males receiving 300 or 1000 mg/kg/day and in females receiving 1000 mg/kg/day. Plasma cholesterol concentrationswere low in males and females receiving 1000 mg/kg/day and plasmacreatinine concentration was high in males receiving 1000 mg/kg/day.
After 13 weeks of treatment, liver and kidney weights were high in both sexes given 1000 mg/kg/day and epididymis weights were slightly low in males given 1000 mg/kg/day. Macroscopic findings after 13 weeks of treatment were confined to the presence of depressions on the non-glandular stomach in one male and one female given 1000 mg/kg/day. 
Treatment-related histopathological changes occurred in the stomach, liver and thyroid gland. In the stomach, hyperplasia of the non-glandular epithelium was seen in males and females given 1000 mg/kg/day and ulceration of the non-glandular epithelium was seen in one high dose male. In the liver, centrilobular hypertrophy was recorded in males and females given 1000 mg/kg/day and in the thyroid gland, follicular cell hypertrophy was seen in two males given 1000 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: see below
Organ:
stomach
liver
kidney
thyroid gland
Treatment related:
yes
Dose response relationship:
yes

Dose (mg/kg bw)

0

 

100

 

300

 

1000

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Mortality

0/10

0/10

0/10

0/10

1/10

0/10

1/10

0/10

No

Clinical signs

vocalisation

 

 

1/10

 

 

1/10

 

 

2/10

 

 

3/10

 

Body weight gain

 

 

 

 

 

 

↓ (13%) (BW↓ 9%)

↓ (12%)

yes

Food consumption

 

 

 

 

 

 

↓ (9% ns)

 

 

Behavioral effects

NTRE

No

Grip strength

 

 

 

 

 

 

Hindlimb ↓ (20%)

 

Yes

Motoractivity

NTRE

No

Haematology

 

 

Large unst cells

 

 

 

Neutr

Large unst cells

 

Eos↓

 

Hct↓ (10%)

Hb↓ (5%)

RBC↓ (4%)

WBC↓ (31%)

Lymph ↓ (31%)

Eos, neutr, baso

Large unst cells

 

WBC↓ (22% ns)

Eos

aPTT ↓ (21%)

 

 

 

 

Clinical biochemistry

 

 

 

Bile ac

(45% ns)

Bile ac↑ (62%)

 

Bile ac↑ (29% ns)

Cholest↓ (14% ns)

 

ALP↑ (36%)

Bile ac↑ (65%)

Creat↑(15%)

Cholest↓ (54%)

TP↓ (4%)

A/G↑ (18%)

ALP↑ (27%)

Bile ac↑ (95%)

Cholest↓ (23%)

 

 

Organ weights (adjusted for BW)

 

 

 

Thymus↓ (19%)

 

 

Brain↓ (4%)

Epidi↓ (8%)

Kidney↑(17%)

Liver↑(32%)

Thymus↓ (12% ns)

Kidney↑(7%)

Liver↑(35%)

Thymus↓ (19%)

 

Macroscopy

NTRE

 

Histopathology

-Liver Centrilobular hypertrophy

-StomachHyperplasia, Epithelial, Nonglandular Region

-Thyroid Hypertrophy, Follicular Cells

 

 

 

 

 

 

 

4/9

 

4/9

 

2/9

 

3/10

 

4/10

 

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

Summary of findings in the stomach for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

300

1000

0

100

300

1000

Depression(s)

0

0

0

1

0

0

0

1

Number of tissues examined

10

10

9

9

10

10

10

10

 

Summary of treatment related findings in the stomach for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

300

1000

0

100

300

1000

Hyperplasia, Epithelium,
Non-glandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

3

0

0

0

3

Slight

0

0

0

2

0

0

0

1

Total

0

0

0

5

0

0

0

4

Number of tissues examined

10

10

9

9

10

10

10

10

 

Summary of treatment related findings in the liver for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

300

1000

0

100

300

1000

Hypertrophy, Centrilobular

 

 

 

 

 

 

 

 

Minimal

0

0

0

4

0

0

0

3

Total

0

0

0

4

0

0

0

3

Number of tissues examined

10

10

9

9

10

10

10

10

 

Conclusions:
In the context of this study, the substance showed was in general well tolerated
The NOAEL is 300 mg/kg/day based on local irritation of the stomach, effects on the liver and thyroid in combination with effects on body weight and white blood cells..
Executive summary:

Three groups of 10/sex Han Wistar rats received the substance at doses of 100, 300 and 1000 mg/kg/day. A similarly constituted control group received the vehicle (1% w/v methylcellulose) at the same volume-dose as the treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by daily visual observation), ophthalmic examination, hematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

Results

The appearance and behavior of the animals, sensory reactivity and motor activity were unaffected by treatment and there were no treatment-related deaths.

At the grip strength investigations in Week 12, there was a trend towards low hind limb grip strength in the males, which was statistically significant at 1000 mg/kg/day.

Body weight gain and food consumption were significantly low in males receiving 1000 mg/kg/day and body weight gain was low in females receiving 1000 mg/kg/day.

There were no treatment-related ophthalmic findings.

At the hematological examination performed in Week 13, there were some reductions of leucocyte parameters which attained statistical significance, particularly in the males receiving 1000 mg/kg/day (low lymphocyte, eosinophil, basophil, monocyte, neutrophil and large unstained cell counts with a consequential decrease of total leucocyte count); there was a similar, but less pronounced effect in the females receiving 1000 mg/kg/day, where lymphocyte, eosinophil and neutrophil counts were low, resulting in a consequential decrease of total leucocyte count. 

At the biochemical examination of the blood plasma, plasma alkaline phosphatase activity was high in males and females receiving 1000 mg/kg/day and bile acid concentrations were high in males receiving 300 or 1000 mg/kg/day and in females receiving 1000 mg/kg/day. Plasma cholesterol concentrationswere low in males and females receiving 1000 mg/kg/day and plasma creatinine concentration was high in males receiving 1000 mg/kg/day.

After 13 weeks of treatment, liver and kidney weights were high in both sexes given 1000 mg/kg/day and epididymis weights were slightly low in males given 1000 mg/kg/day. Macroscopic findings after 13 weeks of treatment were confined to the presence of depressions on the non‑glandular stomach in one male and one female given 1000 mg/kg/day. 

Treatment-related histopathological changes occurred in the stomach, liver and thyroid gland. In the stomach, hyperplasia of the non-glandular epithelium was seen in males and females given 1000 mg/kg/day and ulceration of the non-glandular epithelium was seen in one high dose male. In the liver, centrilobular hypertrophy was recorded in males and females given 1000 mg/kg/day and in the thyroid gland, follicular cell hypertrophy was seen in two males given 1000 mg/kg/day.

Conclusion

It is concluded that the substance was well-tolerated, but at 1000 mg/kg/day caused local irritation to the gastric epithelium and hepatocytic hypertrophy that was likely due to adaptive enzyme induction and secondary to rodent-specific hypertrophic changes in the thyroid. Based on these effects and effects on body weight gain, white blood cells and blood biochemistry, the no-observed-adverse-effect-level (NOAEL) is considered to be 300 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
guideline study under GLP
System:
other: see below
Organ:
stomach
liver
kidney
thyroid gland

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

Justification for classification or non-classification

The available data on toxicity after repeated dose of 2-ethylhexyl benzoate do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.