Registration Dossier

Administrative data

Description of key information

According to the available key study (Manciaux, 2001, GLP, OECD Guideline 407 Method, Klimisch2), the test substance monoconstituent 3,5,5 trimethylhexyl 3,5,5-trimethylhexanoate (RN CAS: 59219-71-5) when administered on rats daily during 28 days by gavage at 0, 100, 300 and 1000 mg/kg/day, induced exacerbation of effects induced by vehicle corn oil on liver (effect induced by the high fat load in liver by administration of the vehicle and the test item). Indeed, some animals of each group (including control) showed hepatic steatosis. This hepatic effect was considered to be pathological and irreversible at the dose of 300 mg/kg/day (due to dysregulation of enzymatic activities and aggravated hepatic steatosis). Hence the No Observed Effect Level was defined at 100 mg/kg/day and the Low Observed Adverse Effect Level at 300 mg/kg/day based on hepatic toxic effect. Considering the dose effect, the severity of the effects which refers to known mechanism of fatty acid. According to the CLP criteria, the substance should not be classified with a STOT.

3 studies were available to estimate the repeated dose toxicity by dermal route. No relevant NOAEL or LOAEL could be defined but similar systemic toxic effects than by oral route were reported plus skin irritation effects that were observed after repeated dermal exposure. The results indicated a  probable hydrolysis of the ester in the skin and after oral route in systemic compartment. The toxic effects observed seems due to the 3,5,5 trimethylhexanoic acid formed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Aug - 3 Sep 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study with acceptable restrictions. The analytical purity of the test substance was not specified; no detailed clinical observations were made; no neurobehavioural tests were performed; prostate and epididymides were not weighed.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted Oct 2008
Deviations:
yes
Remarks:
no detailed clinical observations made; no neurobehavioural tests performed; prostate, testes and epididymides not weighed
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Crl CD(SD) IGS BR, COBS-VAF
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River France, Saint Aubin-lès-Elbeuf, France
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 204 g (males), 169 g (females)
- Fasting period before study: no
- Housing: the animals were housed 2 per cage, according to sex and dose group, in suspended wire-mesh cages (43.0 cm x 21.5 cm x 18.0 cm). A metallic tray containing autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage and the sawdust changed once per week.
- Diet: A04 C pelleted maintenance diet, batch No. 90528 (U.A.R., Villemoisson-sur-Orge, France), ad libitum
- Water: tap water filtered using a 0.22 micron filter, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Aug 2001 To: 2-3 Sep 2001
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: the dosing solutions were made daily by mixing the test substance with the vehicle to the required concentration of 20, 60 and 200 mg/mL and homogenising the solutions using a magnetic stirrer. The solutions were stirred continously during the dosing procedure. Doses were adjusted acording to the most recently recorded body weight of each animal.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 107H1649 (Sigma, Saint-Quentin-Fallavier, France)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
29-30 days
Frequency of treatment:
daily, 7 days/week
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and signs of morbidity, at least once daily for clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 (prior to administration), 8, 15, 22 and 29 (prior to sacrifice)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, based on intake recorded once per week
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 of the study period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: yes, overnight (at least 14 hours)
- How many animals: all the animals in the control group and treatment groups
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes (platelets), total leucocytes, differential leucocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes), reticulocytes, prothrombin time, activated partial thromboplastin time, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28 of the study period
- Animals fasted: yes, overnight (at least 14 hours)
- How many animals: all the animals in the control group and treatment groups
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total protein, albumin, albumin/globulin ratio, cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase

URINALYSIS: Yes
- Time schedule for collection of urine: Day 28 of the study period
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes, overnight (at least 14 hours)
- Parameters examined: volume, pH, specific gravity, proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, sediment (microscopic cytology determining leucoytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells), appearance, colour

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Gross pathology was performed in all animals, including examination of the external surfaces; all orifices; the cranial cavity; the external surfaces of the brain and spinal cord; the thoracic, abdominal and pelvic cavities with associated organs and tissues; the neck with associated organs and tissues. The absolute and relative weight of the following organs was determined in all animals: testes, heart, liver, spleen, adrenal glands, kidney, ovaries, thymus, thyroids with parathyroids. The organs were fixed in 10% neutral formaline.

HISTOPATHOLOGY: Yes. In all animals the following organs were preserved in 10% buffered formalin (except for the eyes and Harderian glands that were fixed in Davidson's fixative, and the testes and epididymides that were preserved in Bouin's fluid), embedded in paraffin wax, sectioned and stained with hematoxylin-eosin: adrenals, aorta, brain, caecum, colon, duodenum, epididymides, oesophagus, eyes with Harderian glands, femoral bone with articulation, heart, ileum, jejenum, kidneys, liver, lungs with bronchi, mandibular lymph nodes, mesenterial lymph nodes, mammary gland, ovaries, pancreas, prostate, pituitary gland, rectum, sublingual and submaxillary salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal chord (cervical, thoracic, lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, tongue, trachea, urinary bladder, uterus with horns and cervix, and vagina. The above-mentioned organs and tissues were examined microscopically in animals of the control group and high-dose group, and animals that died or were killed prematurely. In addition, microscopic examination was performed on all macroscopic lesions and the target organs (liver and kidney) of the animals in the low-and mid-dose groups sacrificed at the scheduled time.
Statistics:
Body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data were analysed for statistical significance. The tests were applied according to the flow chart attached in 'Attached background material'.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was note in 5 males and all females given 1000 mg/kg/day, occasionally or up to the end of
the study, after at least 6 days of treatment. Areas of hair loss, scattered hair and/or diffuse alopecia,
were noted in one female given 100 mg/kg/day, in 3 females of the 300 mg/kg/day group, in 3 males
and 7 females of the high dose group.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female died in the group given 300 mg/kg/day. Four females of the high dose level group was
found dead prematurely. No mortality occurred at 100 mg/kg/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No marked differences were noted in bodyweight gain between control animals and animals given
100 or 300 mg/kg/day. When compared to the mean control value, a lower mean body weight gain
was noted in males and females given 1000 mg/kg/day, during the whole treatment period. This
difference was especially marked in week 1 and correlated with lower food consumption. A body
weight loss was noted in week 4 in both males and females. This effect, correlated with lower food
consumption, was attributed to treatment with the test substance
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
When compared to the mean control values, a higher mean monocyte level was noted with doserelationship
in all treated males and females. however, almost of all individual values remained within
the range of historical data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
When compared to mean control values, the following differences were noted at the end of the
treatment period:
-higher Aspartate aminotransferase and Alanine aminotransferase activities (associated in females
with higher Alkaline Phosphatase activity) in females given 300 mg/kg/day and all animals given 1000
mg/kg/day was measured.
-a lower mean glucose in all treated animals, almost all the individual values remained within the
range of historical data values. This effect could be related to describe below changes observed in
liver.
-a higher urea level in all treated males and females was observed which was in the range of histor
ical data.
-a higher mean inorganic phosphorus level in females given 100 or 300 mg/kg /day and in males an
d females given 1000 mg/kg /day were noted. Due to low control values, the higher mean inorganic
phosphorus values was not considered as toxicological relevant, there was not correlated with ionic
changes and all individual datas were within the historical data.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The only difference noted was a statistically higher volume of urine in females given 100 mg/kg/day,
and in males and females of the high dose group. This was correlated with a lower specific gravity.
The higher urine volume for the high dose level group was considered to be related to the treatement
and could be related to the renal changes observed among these animal (described below).
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Description (incidence and severity):
The higher kidneys weights observed in the males were considered treatment related, could b
e correlated to the acidophilic globules in the cortical tubular epithelium of the kidneys noted at
microscopic examination (section histopathological findings : non-neoplastic section). In females of
the 300 and 1000 mg/kg/day dose groups, a higher kidney weights was observed.Higher dose related liver weights were observed in two sexes.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day: increased kidney weight (males, non-adverse)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Grey/green colouration of the kidneys were noted in one male and one female of the group given 100
mg/kg/day, in two females of the intermediate dose group, in five male and one female of the 1000
mg/kg/day group. This effect was related to males only, with the acidophilic globules in the cortical
tubular epithelium.
Liver enlargement was noted in one male given 100 mg/kg/day, in one male and female given 300 m
g/kg/day, in 6 males and 5 females of the 1000 mg/kg/day dose group. Accentuated lobular pattern
was noted in 4 males and 7 females given 100 mg/kg/day, in 4 males and 9 females given 300 mg/
kg/day, in 8 males and 3 females of the high dose group. Liver paleness was noted in 4 females given
100 mg/kg/day, in 3 females given 300 mg/kg/day, in 2 males and 7 females of the high dose group.
All this effects on the liver were correlated with minimal to severe steatosis observed and/or slight to
severe hepatocellular hypertrophy observed in microscopic examination.
Reduced spleen (1 female of the 300 mg/kg/day group, in 1 female and 4 males of the high d
ose group) ,reduced thymus (2 females given 1000 mg/kg/day) observed were considered to be
secondary to the changes which occured on the liver and kidneys.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal to severe steatosis (perilobular, perilobular and mediolobular, or diffuse)was seen in 5 con
trol females, in 5 males of the 100 mg/kg/day group, 7 males of the intermediate dose group, 9 male
s given 1000 mg/kg/day and all the treated females. Slight to severe hepatocellular hypertrophy was
seen in 9 males of 300 and 1000 mg/kg/day groups and in all females treated with this doses.
Minimal to slight acidophilic globules in cortical tubular epithelium were noted in 3 control males. Sl
ight to severe acidophilic globules in the cortical tubular epithelium were noted in all males of all the
test substance treated groups. The higher incidence and severity of the acidophilic globule was consi
dered to be caused by the increased production of alpha-2-microglobulin. This effect is only observed
in male rodents.
Reduced spleen (1 female of the 300 mg/kg/day group, in 1 female and 4 males of the high dose gro
up) ,reduced thymus (2 females given 1000 mg/kg/day) observed were considered to be secondary to
the changes which occured on the liver and kidneys.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
clinical signs
gross pathology
histopathology: non-neoplastic
mortality
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
clinical signs
histopathology: neoplastic
mortality
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Body weight

 

Group (mg/kg bw/day)

 

Males

Females

Day

Control

100

300

1000

Control

100

300

1000

1

204 ± 11.0

203 ± 11.1

206 ± 10.6

204 ± 9.3

168 ± 9.3

169 ± 8.3

167 ± 6.3

171 ± 8.9

8

263 ± 15.2

259 ± 14.8

261 ± 11.6

243 ± 26.2*

194 ± 10.7

195 ± 10.9

190 ± 8.9

173 ± 16.7**

15

308 ± 19.9

307 ± 20.5

305 ± 14.7

289 ± 30.2

216 ± 13.9

221 ± 17.2

212 ± 9.9

198 ± 28.3

22

337 ± 23.5

332 ± 31.0

329 ±16.5

317 ± 27.3

233 ± 17.5

241 ± 21.2

231 ± 8.6

223 ± 25.8

29

355 ± 26.1

346 ± 36.2

337 ± 19.6

310 ± 29.3**

240 ± 16.8

250 ± 23.5

235 ± 9.3

214 ± 36.6

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 2: Body weight Day 1 - 28

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Total body weight gain (g)

+151

+143

+131

+106

+72

+81

+68

+43

Body weight change Day 1 - 28 (relative)

x 1.7

x 1.7

x 1.6

x 1.5

x 1.4

x 1.5

x 1.4

x 1.3

Difference from control (%)

-

-3

-5

-13

-

-4

-2

-11

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 3: Selected haematology results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Monocytes (G/L)

0.28 ± 0.094 

0.32 ± 0.155

0.46 ± 0.332

0.47 ± 0.258

0.17 ± 0.077

0.19 ± 0.077

0.25 ± 0.104

0.36 ± 0.095**

WBC

10.78 ± 1.97

8.45 ± 2.04

9.84 ± 3.75

10.36 ± 2.07

6.28 ± 1.26

6.74 ± 1.63

7.57 ± 2.30

6.00 ± 1.19

APTT

23.2 ± 3.70

24.7 ± 3.67

24.5 ± 5.80

17.9 ± 4.51*

16.1 ± 2.71

15.4 ± 1.32

14.1 ± 1.43

15.5 ± 3.15

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 4: Selected clinical chemistry results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Calcium (mmol/L)

2.66 ± 0.05

2.78 ± 0.07**

2.75 ± 0.07*

2.74 ± 0.08*

2.77 ± 0.06

2.62 ± 0.06*

2.67 ± 0.09**

2.62 ± 0.08**

Inorganic phosphate (mmol/L)

2.69 ± 0.197

2.69 ± 0.157

2.73 ± 0.100

2.94 ± 0.192**

1.98 ± 0.255

2.30 ± 0.279*

2.41 ± 0.123**

2.58 ± 0.223**

Glucose (mmol/L)

7.17 ± 0.592

5.85 ± 0.436**

5.50 ± 0.190**

5.49 ± 0.684**

7.08 ± 0.806

6.13 ± 0.489*

5.70 ± 0.885**

5.81 ± 1.193*

Urea (mmol/L)

3.5 ± 0.59

4.1 ± 0.59

4.5 ± 0.63**

4.3 ± 0.48*

5.1 ± 0.48

6.0 ± 1.10

6.5 ± 1.60*

6.6 ± 1.02*

ALP (IU/L)

313 ± 35.6

312 ± 62.6

279 ± 51.4

338 ± 111.8

190 ± 40.4

244 ± 73.5

316 ± 118.7**

364 ± 158.9**

ASAT (IU/L)

57 ± 6.3

56 ± 7.2

69 ± 13.9

86 ± 29.1**

59 ± 15.2

50 ± 18.9

75 ± 17.9

120 ± 41.1**

ALAT (IU/L)

14 ± 2.2

18 ± 4.5

21 ± 8.0

42 ± 25.9**

14 ± 5.3

24 ± 10.1

35 ± 12.3**

48 ± 15.8**

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 5: Urinary volume results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Volume (mL)

14 ± 11.2

14 ± 7.7

19 ± 7.8

28 ± 12.0*

11 ± 4.2

23 ± 9.0*

17 ± 14.1

27 ± 13.6*

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 6: Selected organ weights

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Kidney, absolute (g)

2.62 ± 0.249

3.09 ± 0.320*

3.00 ± 0.287

2.98 ± 0.536

1.79 ± 0.168

2.01 ± 0.189*

1.85 ± 0.95

1.91 ± 0.255

Kidney, relative (%)

0.786 ± 0.047

0.965 ± 0.097**

0.964 ± 0.084**

1.03 ± 0.098**

0.801 ± 0.061

0.882 ± 0.062**

0.877 ± 0.043*

0.990 ± 0.052**

Liver, absolute (g)

10.9 ± 1.84

11.26 ± 1.47

13.02 ± 0.820*

16.20 ± 3.60**

7.32 ± 0.750

9.69 ± 1.42*

11.35 ± 1.46**

13.60 ± 4.48**

Liver, relative (%)

3.27 ± 0.385

3.49 ± 0.142

4.19 ± 0.330**

5.62 ± 1.01**

3.27 ± 0.247

4.23 ± 0.309

5.38 ± 0.559**

7.02 ± 2.22**

Spleen, absolute (g)

0.651 ± 0.127

0.626 ± 0.139

0.580 ± 0.117

0.502 ± 0.102*

0.534 ± 0.097

0.538 ± 0.102

0.427 ± 0.060*

0.333 ± 0.115*

Spleen, relative (%)

0.195 ± 0.032

0.194 ± 0.030

0.185 ± 0.029

0.174 ± 0.023

0.239 ± 0.042

0.235 ± 0.033

0.203 ± 0.028

0.170 ± 0.042**

Thymus, absolute (g)

0.469 ± 0.152

0.452 ± 0.104

0.426 ± 0.075

0.360 ± 0.071

0.412 ± 0.058

0.411 ± 0.058

0.328 ± 0.058*

0.260 ± 0.132**

Thymus, relative (%)

0.140 ± 0.042

0.140 ± 0.024

0.137 ± 0.022

0.127 ± 0.030

0.184 ± 0.021

0.180 ± 0.013

0.155 ± 0.021*

0.130 ± 0.050

Ovaries, absolute (g)

-

-

-

-

0.121 ± 0.017

0.129 ± 0.017

0.117 ± 0.012

0.099 ± 0.016*

Ovaries, relative (%)

-

-

-

-

0.054 ± 0.007

0.056 ± 0.006

0.056 ± 0.006

0.051 ± 0.007

Testes, absolute (g)

3.25 ± 0.258

3.15 ± 0.335

3.18 ± 0.193

3.11 ± 0.328

-

-

-

-

Testes, relative (%)

0.978 ± 0.089

0.985 ± 0.107

1.02 ± 0.071

1.09 ± 0.094*

-

-

-

-

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 7: Gross pathology results (incl. deaths/interim sacrifices)

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Liver, accentuated lobular pattern

0/10

4/10

4/10

8/10

0/10

7/10

9/10

3/10

Liver, enlarged

0/10

1/10

1/10

6/10

0/10

0/10

1/10

5/10

Liver, paleness

0/10

0/10

0/10

2/10

0/10

4/10

3/10

7/10

Kidney, grey/green colour

0/10

1/10

3/10

5/10

0/10

1/10

2/10

1/10

Spleen, reduced in size

0/10

0/10

0/10

1/10

0/10

0/10

1/10

4/10

Thymus, reduced in size

0/10

0/10

0/10

0/10

0/10

0/10

0/10

2/10

 

Table 8: Histopathology results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Liver, moderate to severe steatosis

0/10

5/10

7/10

8/10

5/10

10/10

10/10

10/10

Liver, slight to severe hepatocellular hypertrophy

0/10

0/10

9/10

9/10

0/10

0/10

10/10

10/10

Kidney, acidophilic globules in cortical tubular epithelium, minimal to slight

0/10

10/10

10/10

10/10

0/10

0/10

0/10

0/10

Kidney, vacuolated cortical tubular epithelium, minimal to moderate

0/10

0/10

0/10

0/10

0/10

0/10

2/10

6/10

Spleen, contracted

0/10

0/10

0/10

0/10

0/10

0/10

0/10

5/10

Thymus, lymphoid depletion

0/10

0/10

0/10

0/10

0/10

0/10

0/10

7/10

 

Conclusions:
Under the experimental conditions of the study, the test substance induced mortality at 300 and 1000 mg/kg/day, signs of kidney and liver steatosis all dose levels (grading slight to severe steatosis) with hepatocellular hypertrophy (grading slight to severe). This hepatic steatosis observed was present in all groups including control group (5 females) due to high fat load in liver. Indeed the vehicle, corn oil , was used for steatosis model in rats (CIR comments) and the effect noted was related to physiological metabolism of fatty acid in liver. This changes in liver became pathological in case of irreversible dysregulation of metabolism seen in higher activity of Alkaline phosphatase, Alanine transferase acti
vities and lower mean value of plasmatic cholesterol (1000 mg/kg/day dose group). This effects was correlated to higher severity grading of steatosis at this two dose levels. In regard with the steatosis induced by control vehicle, the test item exacerbated the changes induced by corn oil. However, this changes was not adverse at control condition and at 100 mg/kg/day, there was no dysregulation of enzymatic activities and no variation of lipidemy in plasma (seen in plasmatic cholosterol and triglyceride concentration) and did not led to mortality (observed at 300 and 1000 mg/day/kg groups). Lower glycemia was observed in all tested animal and was considered as non adverse because values were within the range of historical data of the laboratory.
The No Observed Adverse Effect NOAEL in rats for the test substance in rat treated by oral route was defined at 100 mg/kg/day, the steatosis at this dose was not considered as pathological and adverse, no enzymatic dysregulation was observed, no higher fatty acid in blood was noted, the changes of liver was not considered as irreversible.
Executive summary:

The purpose of this GLP-compliant study was to evaluate the potential toxicity of the test substance 3,5,5 trimethyl 3,5,5 trimethylhexanoate when daily administered orally in rats during 28 days according to the OECD Guideline 407 method.

Four groups of 20 Sprague Dawley rats (composed with 10 males and 10 females) were used in this repeated dose toxicity test by gavage route. They were exposed to different doses as 0, 100, 300 and 1000 mg/kg/day. The test substance was diluted in corn oil. The control group only received vehicle. During treatment period, different parameters were measured. The mortaility, clinical signs were checked daily. Body weight and food consumption were recorded once a week. Haematological, blood biochemical and urinalysis were performed the last day of the treatment period. On completion of the treatment period, animal were sacrified by CO2 asphyxiation and blood exsanguination. Macroscopic

examination of tissues, with organ weighing and microscopic examination were performed on each animal (on survived animals and animals which died prematurely).

No marked differences were noted in bodyweight gain between control animals and animals given 100 or 300 mg/kg/day. When compared to the mean control value, a lower mean body weight gain was noted in males and females given 1000 mg/kg/day, during the whole treatment period. This difference was especially marked in week 1 and correlated with lower food consumption. A body weight loss was noted in week 4 in both males and females. This effect, correlated with lower food consumption, was attributed to treatment with the test substance.

Ptyalism was note in 5 males and all females given 1000 mg/kg/day, occasionally or up to the end of the study, after at least 6 days of treatment. Areas of hair loss, scattered hair and/or diffuse alopecia, were noted in one female given 100 mg/kg/day, in 3 females of the 300 mg/kg/day group, in 3 males and 7 females of the high dose group.

One female died in the group given 300 mg/kg/day. Four females of the high dose level group was found dead prematurely. No mortality occurred at 100 mg/kg/day.

When compared to mean control values, the following differences were noted at the end of the treatment period:

-higher Aspartate aminotransferase and Alanine aminotransferase activities (associated in females with

higher Alkaline Phosphatase activity) in females given 300 mg/kg/day and all animals given 1000 mg/kg/day was measured.

-a lower mean glucose in all treated animals, almost all the individual values remained within the range of historical data values. This effect could be related to describe below changes observed in liver.

-a higher urea level in all treated males and females was observed which was in the range of historical data.

-a higher mean inorganic phosphorus level in females given 100 or 300 mg/kg /day and in males and females given 1000 mg/kg /day were noted. Due to low control values, the higher mean inorganic phosphorus values was not considered as toxicological relevant, there was not correlated with ionic changes and all individual datas were within the historical data.

Grey/green colouration of the kidneys were noted in one male and one female of the group given 100 mg/kg/day, in two females of the intermediate dose group, in five male and one female of the 1000 mg/kg/day group. This effect was significant for males only, with the acidophilic globules in the cortical tubular epithelium.

Liver enlargement was noted in one male given 100 mg/kg/day, in one male and female given 300 mg/kg/day, in 6 males and 5 females of the 1000 mg/kg/day dose group. Accentuated lobular pattern was noted in 4 males and 7 females given 100 mg/kg/day, in 4 males and 9 females given 300 mg/kg/day, in 8 males and 3 females of the high dose group. Liver paleness was noted in 4 females given 100 mg/kg/day, in 3 females given 300 mg/kg/day, in 2 males and 7 females of the high dose group. All this effects on the liver were correlated with minimal to severe steatosis observed and/or slight to severe hepatocellular hypertrophy observed in microscopic examination.

Reduced spleen (1 female of the 300 mg/kg/day group, in 1 female and 4 males of the high dose group) ,reduced thymus (2 females given 1000 mg/kg/day) observed were considered to be secondary to the changes which occurred on the liver and kidneys.

Under the experimental conditions of the study, the test substance induced mortality at 300 and 1000 mg/kg/day, signs of kidney and liver steatosis all dose levels (grading slight to severe steatosis) with hepatocellular hypertrophy (grading slight to severe). This hepatic steatosis observed was present in all groups including control group (5 females) due to high fat load in liver. Indeed the vehicle, corn oil, was used for steatosis model in rats (CIR comments) and the effect noted was related to physiological metabolism of fatty acid in liver. This changes in liver became pathological in case of irreversible dysregulation of metabolism seen in higher activity of Alkaline phosphatase, Alanine transferase activities and lower mean value of plasmatic cholesterol (1000 mg/kg/day dose group).

This effects was correlated to higher severity grading of steatosis at this two dose levels. In regard with the steatosis induced by control vehicle, the test intem exacerbated the changes induced by corn oil. However, this changes was not adverse at control condition and at 100 mg/kg/day, there was no dysregulation of enzymatic activities and no variation lipidemy in plasma (seen in plasmatic cholosterol and triglyceride concentration) (observed at 300 and 1000 mg/day/kg groups). Lower glycemia was observed in all tested animal and was considered as non adverse because values were within the range of historical data of the laboratory.

The No Observed Adverse Effect NOAEL in rats for the test substance in rat treated by oral route was defined at 100 mg/kg/day, the steatosis at this dose was not considered as pathological and adverse, no enzymatic dysregulation was observed, no higher fatty acid in blood was noted, the changes of liver was not considered as irreversible.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 2) study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 - 25 Aug 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions. The test substance was administered for 8 or 14 days, no dressing was used for the application, the exposure time was 6 hours, the analytical purity of the test substance is not specified.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted May 1981
Deviations:
yes
Remarks:
test substance administered 8 or 14 days, open application, 6-hour exposure time
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl CD(SD) IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River France, Saint-Aubin-lès-Elbeuf, France
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 178-265 g (males), 169-194 g (females)
- Housing: the animals were housed individually in suspended wire-mesh cages (43 cm x 21.5 cm x 18 cm). A metallic tray with autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage.
- Diet: A04 C pelleted manitenance diet, batch No. 90528 (UAR, Villemoisson-sur-Orge, France), ad libitum
- Water: tap water, filtered through a 0.22 micron filter, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 12/hour (filered, non-recycled)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 Aug 1999 To: 18 Aug 1999 (high-dose group), 25 Aug 1999 control group, low-, and mid-dose group)
Type of coverage:
open
Vehicle:
corn oil
Details on exposure:
TEST SITE
- Area of exposure: dorsal area, approximately 40-45 cm² in males and 30-35 cm² in females
- % coverage: 10
- Type of wrap if used: none; the animals wore a protective collar during the 6-hour exposure period until the test substance was removed by washing
- Time intervals for shavings or clippings: the day before the first administration; thereafter at least 4 hours before administration, whenever necessary, but at least once per week

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, the application area was cleaned with water and dried with absorbent paper
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 mL/kg bw
- Constant volume or concentration used: a constant concentration was appplied, ajusted to the latest body weight recorded.

VEHICLE
- Amount(s) applied (volume or weight with unit): 1 mL/kg bw. The highest dose level was applied undiluted, while the low- and mid- doses were diluted with the vehicle.
- Lot/batch no. (if required): 107H1649 (Sigma, Saint-Quentin-Fallavier, France)

USE OF RESTRAINERS FOR PREVENTING INGESTION: no, a collar was put on the animals for the duration of the exposure period
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
15 days (control group, 100 and 300 mg/kg bw/day)
8 days (860 mg/kg bw/day, sacrifice on Day 9)
Frequency of treatment:
Daily, 7 days/week
Remarks:
Doses / Concentrations:
100, 300 and 860 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the highest dose level was selected by multiplying the relative density of 0.86 with the limit dose level of 1000 mg/kg bw/day recommended in OECD guideline 410, to reach a dose level of 860 mg/kg bw/day
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and morbidity; once daily for clinical signs
- Cage side observations included: general observations of behaviour, condition, external changes like wounds

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION (if dermal study): Yes, scored according to the Draize scoring system
- Time schedule for examinations: daily, prior to test substance administration

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 prior to test substance administration, and twice weekly thereafter during the study period

FOOD CONSUMPTION: Food consumption was recorded twice weekly during the study period
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 9 (high-dose group) and Day 14 (control group, low-and mid-dose groups), prior to test substance administration
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes, overnight for at least 14 hours
- How many animals: all animals in the control and treatment groups
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, thrombocytes, white blood cells, differential white cell count with cell morphology, neutrophils, eosinophils, basophils, lymphocytes, monocytes, reticulocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 9 (high-dose group) and Day 14 (control group, low-and mid-dose groups), prior to test substance administration
- Animals fasted: Yes, overnight for at least 14 hours
- How many animals: all animals in the control and treatment groups
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, cholesterol, triglycerides, alkaline phosphatase, asparatate aminotransferase, alanine aminotransferase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Gross pathology was performed in all animals, including examination of the external surfaces; all orifices; the cranial cavity; the external surfaces of the brain and spinal cord; the thoracic, abdominal and pelvic cavities with associated organs and tissues; the neck with associated organs and tissues. The absolute and relative weight of the following organs was determined in all animals: testes, heart, liver, spleen, adrenal glands, kidney, ovaries, thymus, thyroids with parathyroids.

HISTOPATHOLOGY: Yes. In all animals the following organs were preserved in 10% buffered formalin (except for the eyes and Harderian glands that were fixed in Davidson's fixative, and the testes and epididymides, which were preserved in Bouin's fluid): adrenals, aorta, brain, caecum, colon, duodenum, epididymides, oesophagus, eyes with Harderian glands, femoral bone with articulation, heart, ileum, jejenum, kidneys, liver, lungs with bronchi, mandibular lymph nodes, mesenterial lymph nodes, mammary gland, ovaries, pancreas, prostate, pituitary gland, rectum, sublingual and submaxillary salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal chord (cervical, thoracic, lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, tongue, trachea, urinary bladder, uterus with horns and cervix, and vagina. The kidneys, liver and skin (2 samples of treated area and 1 sample of untreated area) were prepared for microscopic examination by embedding in paraffin wax, sectioning and staining with hematoxylin-eosin. The tissue of kidneys, liver and skin were examined microscopically in animals of the control group and all treatment groups. In addition, microscopic examination was performed on the spleen, stomach with forestomach and thymus for animals in the control and high-dose groups, and on the adrenals of the animals in the control, low-, mid- and high-dose groups.
Statistics:
Body weight, food consumption, hematology, clinical chemistry, urinalysis and organ weight data were analysed for statistical significance. The tests were applied according to the flow chart attached in 'Attached background material'. For hematology and clinical chemistry no statistical evaluation was made for the high-dose groups, as the animals were terminated early and there were no corresponding control values. The results were compared with laboratory historical control values from 8-week old rats. No organ weight data were reported for the high-dose group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: erythema, cutaneous lesions
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: very slight to severe erythema, cutaneous lesions
Mortality:
mortality observed, treatment-related
Description (incidence):
860 mg/kg bw/day: erythema, cutaneous lesions
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: reduced body weight
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: reduced food consumption
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: reduced WBC count
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: increased urea; increased ASAT (female) (non adverse)
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No data available for high-dose groups. 300 mg/kg bw/day: increased liver weight (non adverse)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: accentuated lobular pattern and paleness of the liver; skin scabs
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: hepatocellular hypertrophy, steatosis; skin necrosis, acanthosis, ulceration, inflammatory cells; contracted spleen, lymphoid depression of the thymus secondary to poor health
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no mortality during the study period. 1/5 low-dose males had scabs at the application site from Day 11, while 1/5 females in the mid-dose group had very slight erythema at the application site (time of appearance not reported). In the female high-dose group the first case of erythema was observed on Day 4 and in the male high-dose group on Day 6. The number of erythema cases and the severity increased rapidly, with more severe reactions, on average, in females than in males. All the animals in the high-dose group had very slight to severe erythema and cutaneous lesions, including necrosis, at the application site by Day 9 (see Table 1 under 'Any other information on results incl. tables'). The animals were sacrificed on Day 9 due to the serious skin effects.

BODY WEIGHT AND WEIGHT GAIN
The body weight of high-dose males and females was significantly reduced compared with the control group by Day 8 (males: 19%, females: 15%). See Table 2 under 'Any other information on results incl. tables'.

FOOD CONSUMPTION
In males and females of the high-dose group, the food consumption was significantly reduced compared with the control group (males: 17%, females: 14%).

HAEMATOLOGY
For the high-dose group the results are compared with historical control values, as these animals were sacrificed at a different time point from the control group:
In the high-dose group, a slightly low mean white blood cell (WBC) count was noted in the males (9.38 G/L, compared with the historical control of 8.97 - 21.65 G/L) and females (5.09 G/L, compared with the historical control of 5.39 - 16.89 G/L). The mean eosinophil count was low in males (0.03 G/L compared with the historical control of 0.02 - 0.27 G/L) and females (0.03 G/L compared with the historical control of 0.02 - 0.29 G/L), as was the lymphocyte counts in males (4.24 G/L compared with the historical control of 7.20 - 18.62 G/L) and females (3.02 G/L compared with the historical control of 4.39 - 13.81 G/L). These effects are considered to be related to the inflammatory reactions due to the skin effects at the application site. Significant effects in the low- or mid-dose groups, and in one sex only are considered to be incidental.

CLINICAL CHEMISTRY
For the high-dose group the results are compared with historical control values,, as these animals were sacrificed at a different time point from the control group:
In the high-dose group, a high mean urea level was noted in the males (9.8 mmol/L, compared with the historical control of 3.0 - 5.6 mmol/L) and females (12.6 mmol/L, compared with the historical control of 3.92 - 8.2 mmol/L). In the mid-dose females and males, the urea level was significantly increased compared with the control groups (see Table 3 under 'Any other information on results incl. tables'). The effects are considered to be treatment-related and may be related to the adverse effects observed in the liver.
The protein level was slightly decreased in high-dose males (60 g/L, compared with the historical control of 61 - 71 g/L) and females (57 g/L, compared with the historical control of 62 - 76 g/L). Mid-dose females also showed a significant decrease in protein levels. This may be related to the adverse histopathological effects observed in the liver.
The ALP level was increased in high-dose males (786 IU/L, compared with the historical control of 300 - 789 IU/L) and females (658 IU/L, compared with the historical control of 163 - 478 IU/L). The mid-dose females also had a significant increase in ALP levels. The ASAT level was increased in high-dose males (166 IU/L, compared with the historical control of 34 - 112 IU/L) and females (199 IU/L, compared with the historical control of 44 - 94 IU/L). The mid-dose females also had a non-significant increase in ASAT levels (72 - 74 IU/L). Changes in hepatic enzyme levels indicate a higher metabolic activity, and the changes are considered to be related to the histopathological alterations observed in the liver.
The cholesterol level was decreased in in high-dose males (0.9 mmol/L, compared with the historical control of 1.2 - 2.8 mmol/L) and females (0.5 mmol/L, compared with the historical control of 1.1 - 2.7 mmol/L). In mid-dose females a significant decrease in cholesterol levels was observed. Furthermore, the triglyceride level was decreased in in high-dose males (0.13 mmol/L, compared with the historical control of 0.31 - 1.30 mmol/L) and females (0.18 mmol/L, compared with the historical control of 0.23 - 0.87 mmol/L). These effects may generally be related to the treatment with a fatty ester.
Significant effects in the low- or mid-dose groups, or in one sex only are considered to be incidental.

ORGAN WEIGHTS
No organ weights were reported for the high-dose group, due to the early termination of the animals.
A significant increase in relative liver weight in mid-dose males and females was noted, along with a significant increase in absolute liver weight in females of the same group (see Table 4 under 'Any other information on results incl. tables'). This effect is related to the histopathological effects observed (as detailed below). The relative kidney weight was significantly increased in mid-dose males. As the increase was less than 10% and only observed in one sex, it is not considered to be toxicologically relevant. The absolute and relative thymus weight in females in the mid-dose group was non-significantly increased by 21 and 19%, respectively. The toxicological significance of this observation is unclear. The absolute and relative ovary and testes weight was comparable between the control, low- and mid-dose groups, showing that no effects were noted on the weight of reproductive ogans.

GROSS PATHOLOGY
Scabs were noted on the skin at the application site of 1/5 low-dose males and all the animals in the high-dose group (see Table 5 under 'Any other information on results incl. tables'). The liver showed an accentuated lobular pattern in 0/5, 0/5, 1/5 and 5/5 males in the control, low-, mid- and high-dose group, and in 0/5, 1/5, 4/5 and 5/5 females in the control, low-, mid- and high-dose group. In the high-dose group 3/5 males and 4/5 females had a pale liver, while in 1/5 high-dose males the liver also had a friable consistency. The liver effects are treatment-related and consistent with the microscopic findings. The kidneys of 1/5 females in the mid-dose group, and of 3/5 males and of 1/5 females in the high-dose group, respectively, had a grey-green colour. For the males, the observation was correlated with the microscopic findings reported below. In 2/5 high-dose males and 1/5 females the spleen was reduced in size, while the thymus was reduced in size in 1/5 high-dose females. The finding in the spleen and thymus are considered to be secondary to the general poor health of the animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
In all males and females of the high-dose groups ulceration, degeneration or necrosis and acanthosis of the skin was observed at the application site (see Table 6 under 'Any other information on results incl. tables'). Inflammatory cell count in the dermis of all high-dose animals was determined due to the local skin effects of the substance.
5/5 males and 5/5 females in the mid- and high-dose group, respectively, had hepatocellular hypertrophy. This effect is most likely an adaptive effect of the high hepatic load caused by exposure to the test substance (Bär, A. Charateristics and significance of D-tagatose-induced liver enlargement in rats: an interpretative review. Reg Toxicol Pharmacol 1999, 29:S83 - S93). In the liver, minimal to marked steatosis was observed in 0/5, 0/5, 5/5 and 5/5 males in the control-, low-, mid- and high-dose group, and in 1/5, 5/5, 5/5 and 5/5 females in the control-, low-, mid- and high-dose group. The number of cases and the severity increased with increasing dose, indicating a treatment-related effect. Diets high in fatty acids are known to induce steatosis in rats and the effect is therefore considered to be an adaptive rather than an adverse effect (Buettner, R. et al. Defining high-fat-diet rat models: metabolic and molecular effects of different fat types. J Molec Endocrinol 2006, 6: 485-501). The effect is treatment-related, but considered to be toxicologically relevant in the high-dose group only, due to the correlated hepatic findings noted in the necropsy.
In males, acidophilic globules in the cortical tubular epithelium of the kidney were observed in 5/5 rats in all the treatment groups. This effect is considered to be caused by the increased production sex-specific alpha-2-microglobulin as it is only observed in male rodents and is not relevant to humans. The incidence of renal vacuolated cortical tubular epithelium in 1/5 females in the high-dose group is most likely a treatment-related effect (Altunkaynak, M.E. et al. The effects of high-fat diet on the renal structure and morphometric parametric of kidneys in rats. J Anat 2008, 212:845 - 852). Cases of contracted spleen and lymphoid depletion of the thymus noted in 1/5 or 2/5 high-dose males and 1/5 high-dose females, respectively, is considered to be secondary to the poor general health condition of these animals.

HISTORICAL CONTROL DATA (if applicable)
See historical control data listed above.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects; reduced body weight; reduced food consumption; clinical chemistry (increased urea, increased ALP, ASAT); gross pathology (skin, liver, kidney); organ weights (liver); histopathology (liver, skin)
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
860 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local skin effects (erythema, lesions)
Critical effects observed:
not specified

Table 1: Clinical signs, Day 1 – 16 (control, 100 and 300 mg/kg bw/day), Day 1 – 9 (860 mg/kg bw/day)

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

860

Control

100

300

860

Cutaneous lesions, back

0/5

0/5

0/5

4/5

0/5

0/5

0/5

5/5

Desquamation, back

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Scabs, back

0/5

1/5

0/5

0/5

-

-

-

-

Very slight erythema

0/5

0/5

0/5

1/5

0/5

0/5

1/5

4/5

Well-defined erythema

0/5

0/5

0/5

5/5

0/5

0/5

0/5

4/5

Moderate to severe erythema

0/5

0/5

0/5

1/5

-

-

-

-

Severe erythema

0/5

0/5

0/5

3/5

0/5

0/5

0/5

5/5

No clinical history

5/5

4/5

5/5

0/5

5/5

5/5

4/5

0/5

 

Table 2: Body weight (g)

 

Group (mg/kg bw/day)

 

Males

Females

Day

Control

100

300

860

Control

100

300

860

1

252 ± 5.8

256 ± 5.6

246 ± 10.5

237 ± 33.4

179 ± 7.1

179 ± 8.6

178 ± 6.2

183 ± 9.3

4

279 ± 6.1

282 ± 5.8

271 ± 11.2

259 ± 14.8*

193 ± 9.3

181 ± 9.3

189 ± 9.8

186 ± 4.4

8

309 ± 7.6

311 ± 8.5

300 ± 17.4

250 ± 23.7**

205 ± 11.2

199 ± 1.8

203 ± 12.0

175 ± 12.0**

11

326 ± 11.8

329 ± 13.2

316 ± 20.0

-

215 ± 13.4

211 ± 6.4

209 ± 15.4

-

15

329 ± 12.5

324 ± 13.7

310 ± 22.8

-

209 ± 10.0

207 ± 7.0

206 ± 12.8

-

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 3: Selected clinical chemistry results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

860

Control

100

300

860

Calcium (mmol/L)

2.88 ± 0.052

2.91 ± 0.067

2.80 ± 0.093

-

2.83 ± 0.078

2.76 ± 0.070

2.67 ± 0.072**

-

Urea (mmol/L)

4.9 ± 0.83

5.6 ± 0.37

7.5 ± 1.99**

-

6.0 ± 1.51

7.1 ± 0.96

9.7 ± 1.78**

-

ALP (IU/L)

511 ± 121.7

495 ± 79.2

550 ± 88.8

-

292 ± 79.4

339 ± 47.7

480 ± 163.6*

-

ALAT (IU/L)

23 ± 4.0

20 ± 2.9

19 ± 5.9

-

14 ± 2.3

18 ± 12.2

24 ± 10.3

-

Cholesterol

1.3 ± 0.21

1.3 ± 0.15

1.0 ± 0.27

-

1.8 ± 0.39

0.8 ± 0.26**

0.7 ± 0.11**

-

Protein

68 ± 1.6

71 ± 2.3

68 ± 2.8

-

74 ± 2.0

71 ± 2.4

68 ± 4.8*

-

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 4: Selected organ weights

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

860

Control

100

300

860

Kidney, absolute (g)

2.66 ± 0.176

2.67 ± 0.309

2.88 ± 0.227

-

1.73 ± 0.138

1.73 ± 0.090

1.72 ± 0.086

-

Kidney, relative (%)

0.919 ± 0.07

0.913 ± 0.07

1.05 ± 0.08*

-

0.947 ± 0.064

0.959 ± 0.038

0.966 ± 0.058

-

Liver, absolute (g)

9.44 ± 0.985

10.13 ± 1.06

11.04 ± 1.34

-

6.02 ± 0.523

6.92 ± 0.252

7.91 ± 0.849**

-

Liver, relative (%)

3.2 6 ± 0.331

3.47 ± 0.278

4.02 ± 0.327*

-

3.28 ± 0.185

3.85 ± 0.079

4.44 ± 0.454**

-

Thymus, absolute (g)

0.468 ± 0.101

0.455 ± 0.092

0.453 ± 0.048

-

0.409 ± 0.081

0.396 ± 0.079

0.323 ± 0.033

-

Thymus, relative (%)

0.161 ± 0.032

0.156 ± 0.033

0.165 ± 0.021

-

0.222 ± 0.036

0.219 ± 0/037

0.181 ± 0.011

-

Ovaries, absolute (g)

-

-

-

-

0.107 ± 0.016

0.115 ± 0.013

0.108 ± 0.012

-

Ovaries, relative (%)

-

-

-

-

0.059 ± 0.009

0.064 ± 0.005

0.061 ± 0.007

-

Testes, absolute (g)

3.07 ± 0.096

3.03 ± 0.131

2.80 ± 0.346

-

-

-

-

-

Testes, relative (%)

1.06 ± 0.034

1.04 ± 0.085

1.02 ± 0.121

-

-

-

-

-

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 5: Gross pathology results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

860

Control

100

300

860

Liver, accentuated lobular pattern

0/5

0/5

1/5

5/5

0/5

1/5

4/5

4/5

Liver, friable consistency

0/5

0/5

0/5

1/5

0/5

0/5

0/5

0/5

Liver, paleness

0/5

0/5

0/5

3/5

0/5

0/5

0/5

4/5

Kidney, grey/green colour

0/5

0/5

0/5

3/5

0/5

0/5

1/5

1/5

Spleen, reduced in size

0/5

0/5

0/5

2/5

0/5

0/5

0/5

1/5

Thymus, reduced in size

0/5

0/5

0/5

0/5

0/5

0/5

0/5

1/5

Skin, scabs at application site

0/5

1/5

0/5

5/5

0/5

0/5

0/5

5/5

 

Table 6: Histopathology results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

860

Control

100

300

860

Liver, minimal to marked steatosis

0/5

0/5

5/5

5/5

1/5

5/5

5/5

5/5

Liver, minimal to moderate hepatocellular hypertrophy

0/5

0/5

5/5

5/5

0/5

0/5

5/5

5/5

Kidney, acidophilic globules in cortical tubular epithelium, minimal to slight

0/5

5/5

5/5

5/5

0/5

0/5

0/5

0/5

Kidney, vacuolated cortical tubular epithelium

0/5

0/5

0/5

0/5

0/5

0/5

0/5

1/5

Adrenal glands, cortical cell hypertrophy

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Spleen, contracted

0/5

0/5

0/5

2/5

0/5

0/5

0/5

1/5

Thymus, lymphoid depletion

0/5

0/5

0/5

1/5

0/5

0/5

0/5

1/5

Skin, degenerated/ necrotic epidermis

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Skin, ulceration

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Skin, acanthosis

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Skin, hyperkeratosis

0/5

0/5

0/5

0/5

0/5

0/5

0/5

2/5

Skin, mixed inflammatory cells in the dermis

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 2) study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 - 25 Aug 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions. The test substance was administered for 8 or 14 days, no dressing was used for the application, the exposure time was 6 hours, the analytical purity of the test substance is not specified.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted May 1981
Deviations:
yes
Remarks:
test substance administered 8 or 14 days, open application, 6-hour exposure time
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl CD(SD) IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River France, Saint-Aubin-lès-Elbeuf, France
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 178-265 g (males), 169-194 g (females)
- Housing: the animals were housed individually in suspended wire-mesh cages (43 cm x 21.5 cm x 18 cm). A metallic tray with autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage.
- Diet: A04 C pelleted manitenance diet, batch No. 90528 (UAR, Villemoisson-sur-Orge, France), ad libitum
- Water: tap water, filtered through a 0.22 micron filter, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 12/hour (filered, non-recycled)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 Aug 1999 To: 18 Aug 1999 (high-dose group), 25 Aug 1999 control group, low-, and mid-dose group)
Type of coverage:
open
Vehicle:
corn oil
Details on exposure:
TEST SITE
- Area of exposure: dorsal area, approximately 40-45 cm² in males and 30-35 cm² in females
- % coverage: 10
- Type of wrap if used: none; the animals wore a protective collar during the 6-hour exposure period until the test substance was removed by washing
- Time intervals for shavings or clippings: the day before the first administration; thereafter at least 4 hours before administration, whenever necessary, but at least once per week

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, the application area was cleaned with water and dried with absorbent paper
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 mL/kg bw
- Constant volume or concentration used: a constant concentration was appplied, ajusted to the latest body weight recorded.

VEHICLE
- Amount(s) applied (volume or weight with unit): 1 mL/kg bw. The highest dose level was applied undiluted, while the low- and mid- doses were diluted with the vehicle.
- Lot/batch no. (if required): 107H1649 (Sigma, Saint-Quentin-Fallavier, France)

USE OF RESTRAINERS FOR PREVENTING INGESTION: no, a collar was put on the animals for the duration of the exposure period
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
15 days (control group, 100 and 300 mg/kg bw/day)
8 days (860 mg/kg bw/day, sacrifice on Day 9)
Frequency of treatment:
Daily, 7 days/week
Remarks:
Doses / Concentrations:
100, 300 and 860 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the highest dose level was selected by multiplying the relative density of 0.86 with the limit dose level of 1000 mg/kg bw/day recommended in OECD guideline 410, to reach a dose level of 860 mg/kg bw/day
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and morbidity; once daily for clinical signs
- Cage side observations included: general observations of behaviour, condition, external changes like wounds

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION (if dermal study): Yes, scored according to the Draize scoring system
- Time schedule for examinations: daily, prior to test substance administration

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 prior to test substance administration, and twice weekly thereafter during the study period

FOOD CONSUMPTION: Food consumption was recorded twice weekly during the study period
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 9 (high-dose group) and Day 14 (control group, low-and mid-dose groups), prior to test substance administration
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes, overnight for at least 14 hours
- How many animals: all animals in the control and treatment groups
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, thrombocytes, white blood cells, differential white cell count with cell morphology, neutrophils, eosinophils, basophils, lymphocytes, monocytes, reticulocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 9 (high-dose group) and Day 14 (control group, low-and mid-dose groups), prior to test substance administration
- Animals fasted: Yes, overnight for at least 14 hours
- How many animals: all animals in the control and treatment groups
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, cholesterol, triglycerides, alkaline phosphatase, asparatate aminotransferase, alanine aminotransferase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Gross pathology was performed in all animals, including examination of the external surfaces; all orifices; the cranial cavity; the external surfaces of the brain and spinal cord; the thoracic, abdominal and pelvic cavities with associated organs and tissues; the neck with associated organs and tissues. The absolute and relative weight of the following organs was determined in all animals: testes, heart, liver, spleen, adrenal glands, kidney, ovaries, thymus, thyroids with parathyroids.

HISTOPATHOLOGY: Yes. In all animals the following organs were preserved in 10% buffered formalin (except for the eyes and Harderian glands that were fixed in Davidson's fixative, and the testes and epididymides, which were preserved in Bouin's fluid): adrenals, aorta, brain, caecum, colon, duodenum, epididymides, oesophagus, eyes with Harderian glands, femoral bone with articulation, heart, ileum, jejenum, kidneys, liver, lungs with bronchi, mandibular lymph nodes, mesenterial lymph nodes, mammary gland, ovaries, pancreas, prostate, pituitary gland, rectum, sublingual and submaxillary salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal chord (cervical, thoracic, lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, tongue, trachea, urinary bladder, uterus with horns and cervix, and vagina. The kidneys, liver and skin (2 samples of treated area and 1 sample of untreated area) were prepared for microscopic examination by embedding in paraffin wax, sectioning and staining with hematoxylin-eosin. The tissue of kidneys, liver and skin were examined microscopically in animals of the control group and all treatment groups. In addition, microscopic examination was performed on the spleen, stomach with forestomach and thymus for animals in the control and high-dose groups, and on the adrenals of the animals in the control, low-, mid- and high-dose groups.
Statistics:
Body weight, food consumption, hematology, clinical chemistry, urinalysis and organ weight data were analysed for statistical significance. The tests were applied according to the flow chart attached in 'Attached background material'. For hematology and clinical chemistry no statistical evaluation was made for the high-dose groups, as the animals were terminated early and there were no corresponding control values. The results were compared with laboratory historical control values from 8-week old rats. No organ weight data were reported for the high-dose group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: erythema, cutaneous lesions
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: very slight to severe erythema, cutaneous lesions
Mortality:
mortality observed, treatment-related
Description (incidence):
860 mg/kg bw/day: erythema, cutaneous lesions
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: reduced body weight
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: reduced food consumption
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: reduced WBC count
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: increased urea; increased ASAT (female) (non adverse)
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No data available for high-dose groups. 300 mg/kg bw/day: increased liver weight (non adverse)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: accentuated lobular pattern and paleness of the liver; skin scabs
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
860 mg/kg bw/day: hepatocellular hypertrophy, steatosis; skin necrosis, acanthosis, ulceration, inflammatory cells; contracted spleen, lymphoid depression of the thymus secondary to poor health
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no mortality during the study period. 1/5 low-dose males had scabs at the application site from Day 11, while 1/5 females in the mid-dose group had very slight erythema at the application site (time of appearance not reported). In the female high-dose group the first case of erythema was observed on Day 4 and in the male high-dose group on Day 6. The number of erythema cases and the severity increased rapidly, with more severe reactions, on average, in females than in males. All the animals in the high-dose group had very slight to severe erythema and cutaneous lesions, including necrosis, at the application site by Day 9 (see Table 1 under 'Any other information on results incl. tables'). The animals were sacrificed on Day 9 due to the serious skin effects.

BODY WEIGHT AND WEIGHT GAIN
The body weight of high-dose males and females was significantly reduced compared with the control group by Day 8 (males: 19%, females: 15%). See Table 2 under 'Any other information on results incl. tables'.

FOOD CONSUMPTION
In males and females of the high-dose group, the food consumption was significantly reduced compared with the control group (males: 17%, females: 14%).

HAEMATOLOGY
For the high-dose group the results are compared with historical control values, as these animals were sacrificed at a different time point from the control group:
In the high-dose group, a slightly low mean white blood cell (WBC) count was noted in the males (9.38 G/L, compared with the historical control of 8.97 - 21.65 G/L) and females (5.09 G/L, compared with the historical control of 5.39 - 16.89 G/L). The mean eosinophil count was low in males (0.03 G/L compared with the historical control of 0.02 - 0.27 G/L) and females (0.03 G/L compared with the historical control of 0.02 - 0.29 G/L), as was the lymphocyte counts in males (4.24 G/L compared with the historical control of 7.20 - 18.62 G/L) and females (3.02 G/L compared with the historical control of 4.39 - 13.81 G/L). These effects are considered to be related to the inflammatory reactions due to the skin effects at the application site. Significant effects in the low- or mid-dose groups, and in one sex only are considered to be incidental.

CLINICAL CHEMISTRY
For the high-dose group the results are compared with historical control values,, as these animals were sacrificed at a different time point from the control group:
In the high-dose group, a high mean urea level was noted in the males (9.8 mmol/L, compared with the historical control of 3.0 - 5.6 mmol/L) and females (12.6 mmol/L, compared with the historical control of 3.92 - 8.2 mmol/L). In the mid-dose females and males, the urea level was significantly increased compared with the control groups (see Table 3 under 'Any other information on results incl. tables'). The effects are considered to be treatment-related and may be related to the adverse effects observed in the liver.
The protein level was slightly decreased in high-dose males (60 g/L, compared with the historical control of 61 - 71 g/L) and females (57 g/L, compared with the historical control of 62 - 76 g/L). Mid-dose females also showed a significant decrease in protein levels. This may be related to the adverse histopathological effects observed in the liver.
The ALP level was increased in high-dose males (786 IU/L, compared with the historical control of 300 - 789 IU/L) and females (658 IU/L, compared with the historical control of 163 - 478 IU/L). The mid-dose females also had a significant increase in ALP levels. The ASAT level was increased in high-dose males (166 IU/L, compared with the historical control of 34 - 112 IU/L) and females (199 IU/L, compared with the historical control of 44 - 94 IU/L). The mid-dose females also had a non-significant increase in ASAT levels (72 - 74 IU/L). Changes in hepatic enzyme levels indicate a higher metabolic activity, and the changes are considered to be related to the histopathological alterations observed in the liver.
The cholesterol level was decreased in in high-dose males (0.9 mmol/L, compared with the historical control of 1.2 - 2.8 mmol/L) and females (0.5 mmol/L, compared with the historical control of 1.1 - 2.7 mmol/L). In mid-dose females a significant decrease in cholesterol levels was observed. Furthermore, the triglyceride level was decreased in in high-dose males (0.13 mmol/L, compared with the historical control of 0.31 - 1.30 mmol/L) and females (0.18 mmol/L, compared with the historical control of 0.23 - 0.87 mmol/L). These effects may generally be related to the treatment with a fatty ester.
Significant effects in the low- or mid-dose groups, or in one sex only are considered to be incidental.

ORGAN WEIGHTS
No organ weights were reported for the high-dose group, due to the early termination of the animals.
A significant increase in relative liver weight in mid-dose males and females was noted, along with a significant increase in absolute liver weight in females of the same group (see Table 4 under 'Any other information on results incl. tables'). This effect is related to the histopathological effects observed (as detailed below). The relative kidney weight was significantly increased in mid-dose males. As the increase was less than 10% and only observed in one sex, it is not considered to be toxicologically relevant. The absolute and relative thymus weight in females in the mid-dose group was non-significantly increased by 21 and 19%, respectively. The toxicological significance of this observation is unclear. The absolute and relative ovary and testes weight was comparable between the control, low- and mid-dose groups, showing that no effects were noted on the weight of reproductive ogans.

GROSS PATHOLOGY
Scabs were noted on the skin at the application site of 1/5 low-dose males and all the animals in the high-dose group (see Table 5 under 'Any other information on results incl. tables'). The liver showed an accentuated lobular pattern in 0/5, 0/5, 1/5 and 5/5 males in the control, low-, mid- and high-dose group, and in 0/5, 1/5, 4/5 and 5/5 females in the control, low-, mid- and high-dose group. In the high-dose group 3/5 males and 4/5 females had a pale liver, while in 1/5 high-dose males the liver also had a friable consistency. The liver effects are treatment-related and consistent with the microscopic findings. The kidneys of 1/5 females in the mid-dose group, and of 3/5 males and of 1/5 females in the high-dose group, respectively, had a grey-green colour. For the males, the observation was correlated with the microscopic findings reported below. In 2/5 high-dose males and 1/5 females the spleen was reduced in size, while the thymus was reduced in size in 1/5 high-dose females. The finding in the spleen and thymus are considered to be secondary to the general poor health of the animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
In all males and females of the high-dose groups ulceration, degeneration or necrosis and acanthosis of the skin was observed at the application site (see Table 6 under 'Any other information on results incl. tables'). Inflammatory cell count in the dermis of all high-dose animals was determined due to the local skin effects of the substance.
5/5 males and 5/5 females in the mid- and high-dose group, respectively, had hepatocellular hypertrophy. This effect is most likely an adaptive effect of the high hepatic load caused by exposure to the test substance (Bär, A. Charateristics and significance of D-tagatose-induced liver enlargement in rats: an interpretative review. Reg Toxicol Pharmacol 1999, 29:S83 - S93). In the liver, minimal to marked steatosis was observed in 0/5, 0/5, 5/5 and 5/5 males in the control-, low-, mid- and high-dose group, and in 1/5, 5/5, 5/5 and 5/5 females in the control-, low-, mid- and high-dose group. The number of cases and the severity increased with increasing dose, indicating a treatment-related effect. Diets high in fatty acids are known to induce steatosis in rats and the effect is therefore considered to be an adaptive rather than an adverse effect (Buettner, R. et al. Defining high-fat-diet rat models: metabolic and molecular effects of different fat types. J Molec Endocrinol 2006, 6: 485-501). The effect is treatment-related, but considered to be toxicologically relevant in the high-dose group only, due to the correlated hepatic findings noted in the necropsy.
In males, acidophilic globules in the cortical tubular epithelium of the kidney were observed in 5/5 rats in all the treatment groups. This effect is considered to be caused by the increased production sex-specific alpha-2-microglobulin as it is only observed in male rodents and is not relevant to humans. The incidence of renal vacuolated cortical tubular epithelium in 1/5 females in the high-dose group is most likely a treatment-related effect (Altunkaynak, M.E. et al. The effects of high-fat diet on the renal structure and morphometric parametric of kidneys in rats. J Anat 2008, 212:845 - 852). Cases of contracted spleen and lymphoid depletion of the thymus noted in 1/5 or 2/5 high-dose males and 1/5 high-dose females, respectively, is considered to be secondary to the poor general health condition of these animals.

HISTORICAL CONTROL DATA (if applicable)
See historical control data listed above.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects; reduced body weight; reduced food consumption; clinical chemistry (increased urea, increased ALP, ASAT); gross pathology (skin, liver, kidney); organ weights (liver); histopathology (liver, skin)
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
860 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local skin effects (erythema, lesions)
Critical effects observed:
not specified

Table 1: Clinical signs, Day 1 – 16 (control, 100 and 300 mg/kg bw/day), Day 1 – 9 (860 mg/kg bw/day)

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

860

Control

100

300

860

Cutaneous lesions, back

0/5

0/5

0/5

4/5

0/5

0/5

0/5

5/5

Desquamation, back

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Scabs, back

0/5

1/5

0/5

0/5

-

-

-

-

Very slight erythema

0/5

0/5

0/5

1/5

0/5

0/5

1/5

4/5

Well-defined erythema

0/5

0/5

0/5

5/5

0/5

0/5

0/5

4/5

Moderate to severe erythema

0/5

0/5

0/5

1/5

-

-

-

-

Severe erythema

0/5

0/5

0/5

3/5

0/5

0/5

0/5

5/5

No clinical history

5/5

4/5

5/5

0/5

5/5

5/5

4/5

0/5

 

Table 2: Body weight (g)

 

Group (mg/kg bw/day)

 

Males

Females

Day

Control

100

300

860

Control

100

300

860

1

252 ± 5.8

256 ± 5.6

246 ± 10.5

237 ± 33.4

179 ± 7.1

179 ± 8.6

178 ± 6.2

183 ± 9.3

4

279 ± 6.1

282 ± 5.8

271 ± 11.2

259 ± 14.8*

193 ± 9.3

181 ± 9.3

189 ± 9.8

186 ± 4.4

8

309 ± 7.6

311 ± 8.5

300 ± 17.4

250 ± 23.7**

205 ± 11.2

199 ± 1.8

203 ± 12.0

175 ± 12.0**

11

326 ± 11.8

329 ± 13.2

316 ± 20.0

-

215 ± 13.4

211 ± 6.4

209 ± 15.4

-

15

329 ± 12.5

324 ± 13.7

310 ± 22.8

-

209 ± 10.0

207 ± 7.0

206 ± 12.8

-

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 3: Selected clinical chemistry results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

860

Control

100

300

860

Calcium (mmol/L)

2.88 ± 0.052

2.91 ± 0.067

2.80 ± 0.093

-

2.83 ± 0.078

2.76 ± 0.070

2.67 ± 0.072**

-

Urea (mmol/L)

4.9 ± 0.83

5.6 ± 0.37

7.5 ± 1.99**

-

6.0 ± 1.51

7.1 ± 0.96

9.7 ± 1.78**

-

ALP (IU/L)

511 ± 121.7

495 ± 79.2

550 ± 88.8

-

292 ± 79.4

339 ± 47.7

480 ± 163.6*

-

ALAT (IU/L)

23 ± 4.0

20 ± 2.9

19 ± 5.9

-

14 ± 2.3

18 ± 12.2

24 ± 10.3

-

Cholesterol

1.3 ± 0.21

1.3 ± 0.15

1.0 ± 0.27

-

1.8 ± 0.39

0.8 ± 0.26**

0.7 ± 0.11**

-

Protein

68 ± 1.6

71 ± 2.3

68 ± 2.8

-

74 ± 2.0

71 ± 2.4

68 ± 4.8*

-

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 4: Selected organ weights

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

860

Control

100

300

860

Kidney, absolute (g)

2.66 ± 0.176

2.67 ± 0.309

2.88 ± 0.227

-

1.73 ± 0.138

1.73 ± 0.090

1.72 ± 0.086

-

Kidney, relative (%)

0.919 ± 0.07

0.913 ± 0.07

1.05 ± 0.08*

-

0.947 ± 0.064

0.959 ± 0.038

0.966 ± 0.058

-

Liver, absolute (g)

9.44 ± 0.985

10.13 ± 1.06

11.04 ± 1.34

-

6.02 ± 0.523

6.92 ± 0.252

7.91 ± 0.849**

-

Liver, relative (%)

3.2 6 ± 0.331

3.47 ± 0.278

4.02 ± 0.327*

-

3.28 ± 0.185

3.85 ± 0.079

4.44 ± 0.454**

-

Thymus, absolute (g)

0.468 ± 0.101

0.455 ± 0.092

0.453 ± 0.048

-

0.409 ± 0.081

0.396 ± 0.079

0.323 ± 0.033

-

Thymus, relative (%)

0.161 ± 0.032

0.156 ± 0.033

0.165 ± 0.021

-

0.222 ± 0.036

0.219 ± 0/037

0.181 ± 0.011

-

Ovaries, absolute (g)

-

-

-

-

0.107 ± 0.016

0.115 ± 0.013

0.108 ± 0.012

-

Ovaries, relative (%)

-

-

-

-

0.059 ± 0.009

0.064 ± 0.005

0.061 ± 0.007

-

Testes, absolute (g)

3.07 ± 0.096

3.03 ± 0.131

2.80 ± 0.346

-

-

-

-

-

Testes, relative (%)

1.06 ± 0.034

1.04 ± 0.085

1.02 ± 0.121

-

-

-

-

-

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 5: Gross pathology results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

860

Control

100

300

860

Liver, accentuated lobular pattern

0/5

0/5

1/5

5/5

0/5

1/5

4/5

4/5

Liver, friable consistency

0/5

0/5

0/5

1/5

0/5

0/5

0/5

0/5

Liver, paleness

0/5

0/5

0/5

3/5

0/5

0/5

0/5

4/5

Kidney, grey/green colour

0/5

0/5

0/5

3/5

0/5

0/5

1/5

1/5

Spleen, reduced in size

0/5

0/5

0/5

2/5

0/5

0/5

0/5

1/5

Thymus, reduced in size

0/5

0/5

0/5

0/5

0/5

0/5

0/5

1/5

Skin, scabs at application site

0/5

1/5

0/5

5/5

0/5

0/5

0/5

5/5

 

Table 6: Histopathology results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

860

Control

100

300

860

Liver, minimal to marked steatosis

0/5

0/5

5/5

5/5

1/5

5/5

5/5

5/5

Liver, minimal to moderate hepatocellular hypertrophy

0/5

0/5

5/5

5/5

0/5

0/5

5/5

5/5

Kidney, acidophilic globules in cortical tubular epithelium, minimal to slight

0/5

5/5

5/5

5/5

0/5

0/5

0/5

0/5

Kidney, vacuolated cortical tubular epithelium

0/5

0/5

0/5

0/5

0/5

0/5

0/5

1/5

Adrenal glands, cortical cell hypertrophy

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Spleen, contracted

0/5

0/5

0/5

2/5

0/5

0/5

0/5

1/5

Thymus, lymphoid depletion

0/5

0/5

0/5

1/5

0/5

0/5

0/5

1/5

Skin, degenerated/ necrotic epidermis

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Skin, ulceration

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Skin, acanthosis

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Skin, hyperkeratosis

0/5

0/5

0/5

0/5

0/5

0/5

0/5

2/5

Skin, mixed inflammatory cells in the dermis

0/5

0/5

0/5

5/5

0/5

0/5

0/5

5/5

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1.76 mg/cm²
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 2) study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Additional information

Repeated dose toxicity, oral, subacute

CAS 59219-71-5

A 28-day oral repeated dose toxicity study was performed under GLP conditions and similar to OECD 407, using 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate (Manciaux, 2001). 10 Sprague-Dawley rats/sex/dose were administered 100, 300 and 1000 mg/kg bw/day by gavage, for 28 consecutive days. No neurobehavioural tests were performed.

4/10 females in the high-dose group died or were killed prematurely (Day 9 - 25). Three of the four animals showed signs of poor clinical condition prior to death, like emaciation, locomotor difficulties, dyspnea and coldness to the touch. The post-mortem examination showed adaptive liver effects, contracted spleen, and vacuolated cortical tubular epithelium in the kidneys in at least 3/4 females. Although no direct cause of mortality/ moribund condition could be found, the general poor condition and post-mortem findings are considered to be toxicologically relevant. The death of 1/10 females in the mid-dose group on Day 11 is considered to be treatment-related. In males administered the highest dose, the body weight was significantly lower on Day 8 and Day 29 than in the control group. In females of the high-dose group, the lower body weight was significant on Day 8, and marked on Day 29 (-11%). The females that died or were sacrificed prematurely were reported to be emaciated. The body weight gain was reduced in males (significantly) and females (non-significantly) during the whole study period, compared with the body weight gain of the control group. The significant effects on body weight (gain) in the high-dose group are considered to be toxicologically relevant. The food intake was significantly reduced in the high-dose groups during week 1 only, compared with the control group. An increase in monocyte level in the high-dose males (non-significant) and females (significant) was observed. This is considered not to be toxicologically relevant, as the mean values were within the historical data range of 0.11 - 0.83 G/L for males and 0.07 - 0.58 G/L for females, and there were no effects on other individual lymphocyte counts, or on the total lymphocyte count. Other significant effects observed only in one sex, or in the low- or mid- dose group only are viewed as incidental occurrences, due to a lack of dose-reponse relation. The alanine aminotransferase (ALAT) levels were increased non-significantly in the mid-dose group and significantly in the high-dose group in males and females. The aspartate aminotransferase (ASAT) levels were increased non-significantly in the mid-dose males, and significantly in the mid-dose females and the high-dose group in males and females. A significant increase in the alkaline phosphatase (ALP) level was noted in high-dose females only. The increased activity of these hepatic enzymes indicates an adaptive increase in hepatic metabolism caused by the treatment and is also related to the pathological changes observed in the liver. Effects noted at the mid-dose level are not considered to be relevant due to non-significance or effects in one sex only. The glucose level was significantly decreased at all dose levels in males and females. According to the study report, almost all the individual values fell within the historical control range of the laboratory (5.45 - 9.84 mmol/L for males and 4.81 - 8.90 mmol/L for females), however the individual results were not included in the study report. In addition, taking into consideration that the dose-related effect is considered to be related to the adaptive liver changes, the decrease in glucose level is not considered as toxicologically relevant. Significant increases in the urea levels were noted in males and females in the mid-and high-dose groups. According to the study report almost all the individual values fell within the historical control range of the laboratory (3.4 - 7.3 mmol/L for males and 3.9 - 8.0 mmol/L for females), however the individual results were not included in the study report. However, the dose-related effect is considered to be toxicologically relevant at the highest dose level only, as histopathological effects were noted in the kidneys at this dose level. A significant increase in the inorganic phosphatase level was noted in the low- mid- and high dose females, as well as in the high-dose males. However, the control value in the females was low compared with the historical control values from the laboratory, and all the individual values fell within the historical control range of the laboratory (2.40 - 3.25 mmol/L for males and 1.66 - 2.77 mmol/L for females), leading to the conclusion that this is not a toxicologically relevant effect. The calcium levels were increased significantly in all dose groups in males and females. However, this is not considered to be of toxicological significance as the increases were less than 10%.

A significant increase in urine volume in the low-dose females is most likely incidental due to the lack of similar effects in the mid-dose animals, while the significant increase observed in the high-dose animals is considered to be toxicologically relevant. 

A dose-related increase in absolute and relative liver weight was observed at all dose levels compared with the control groups, with significant difference from the control group in the mid- and high dose groups for males and females. The increase is considered to be treatment-related, most likely due to adaptive processes based on increased hepatic metabolism, and correlated with macroscopic and microscopic changes. The relative kidney weight was significantly increased compared with the controls at all dose levels for males and females. The effects noted in males at all dose levels are considered to be related to the male rat-specific histopathological findings, while for females a possible histopathological (treatment- but not toxicologically relevant) relation was noted at the mid- and high dose levels. Reduced spleen weight and thymus weight noted in high-dose females were related to the general poor health condition of some individuals in this group. The decrease in absolute ovary weight in high-dose females and increase in relative testes weight in high-dose males compared with the control groups, respectively, is considered to be incidental as the changes were less than 10%.

In the macroscopic examination, a grey/green colouration of the kidney was noted in 0/10, 1/10, 3/10 and 5/10 males in the control, low-, mid- and high-dose group, respectively, and in 0/10, 1/10, 2/10 and 1/10 females in the control, low-, mid- and high-dose group, respectively. A dose-related increase in the number of animals with enlarged liver was observed, while accentuated lobular pattern was reported in 0/10, 4/10, 4/10 and 8/10 males, and 0/10, 7/10, 9/10 and 3/10 females in the control, low-, mid- and high-dose group, respectively. The incidence of liver paleness was notable in females, with 0/10, 4/10, 3/10 and 7/10 in the control, low-, mid- and high-dose group, respectively, while it was observed in 2/10 males in the high-dose group. The reduced spleen- and thymus size noted in high-dose females is considered to be secondary to the poor general health condition of some of these animals.

The microscopic examination showed steatosis in 5/10 females in the control group and in a majority of male and female rats at all dose levels. The number of cases and the severity increased with increasing dose, indicating a treatment-related effect. Diets high in fatty acids are known to induce steatosis in rats and the effect (including the effects on liver weight and gross pathology) is therefore considered to be an adaptive rather than an adverse effect (Buettner, R. et al. 2006). Hepatocellular hypertrophy was noted in 0/10, 0/10, 9/10 and 9/10 males in the control-, low-, mid- and high-dose group, respectively, and in 0/10, 0/10, 10/10 and 10/10 females in the control-, low-, mid- and high-dose group, respectively. This is most likely an adaptive effect of the high hepatic load caused by exposure to the test substance (Bär, A. 1999). In males, acidophilic globules in the cortical tubular epithelium of the kidney was observed in 10/10 rats in all the dose groups. The incidence and severity is considered to be caused by the increased production of alpha-2-microglobulin. This effect is only observed in male rodents and is not relevant to humans. The incidence of renal vacuolated cortical tubular epithelium in 2/10 female rats in the mid-dose group and 6/10 in the high-dose group is a treatment-related effect that is known to occur in rats fed high-fat diets (Altunkaynak, M.E. et al. 2008). Cases of contracted spleen and lymphoid depletion of the thymus noted in high-dose females is considered to be secondary to the poor general health condition of some of the animals.    

Based on the toxicologically relevant effects observed at the highest dose level, the NOAEL is considered to be 100 mg/kg bw/day.

 

Repeated dose toxicity, dermal, subacute

CAS 59219-71-5

A 14-day dermal repeated dose toxicity study was performed under GLP conditions and similar to OECD 410, using 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate (Manciaux, 2001). 5 Sprague-Dawley rats/sex/dose were exposed to 100, 300 and 860 mg/kg bw/day by dermal application, for 14 consecutive days. The test substance was applied undiluted or in corn oil to the shaved skin for 6 hour, open exposure, and the animals wore collars to stop them from grooming and ingesting the substance. The application site was washed with water and dried after each daily exposure. 

There was no mortality during the study period. 1/5 low-dose males had scabs at the application site from Day 11, while 1/5 females in the mid-dose group had very slight erythema at the application site. Starting on Day 4 (females) and 6 (males), all the animals in the high-dose group had very slight to severe erythema and cutaneous lesions, including necrosis, at the application site by Day 9. The number of erythema cases and the severity increased rapidly, with more severe reactions, on average, in females than in males. The animals were sacrificed on Day 9 due to the serious skin irritating effects. The body weight of high-dose males and females was significantly reduced compared with the control group by Day 8 (males: 19%, females: 15%). In males and females of the high-dose group, the food consumption was significantly reduced compared with the control group (males: 17%, females: 14%).

For the high-dose group the hematology and clinical chemistry results are compared with historical control values, as these animals were sacrificed at a different time point from the control group. In the high-dose group, a slightly low mean white blood cell count was noted in the males (9.38 G/L, compared with the historical control of 8.97 - 21.65 G/L) and females (5.09 G/L, compared with the historical control of 5.39 - 16.89 G/L). The mean eosinophil count was low in males (0.03 G/L compared with the historical control of 0.02 - 0.27 G/L) and females (0.03 G/L compared with the historical control of 0.02 - 0.29 G/L), as was the lymphocyte counts in males (4.24 G/L compared with the historical control of 7.20 - 18.62 G/L) and females (3.02 G/L compared with the historical control of 4.39 - 13.81 G/L). These effects are considered to be related to the inflammatory reactions due to the skin effects at the application site.

 

In the high-dose group, a high mean urea level was noted in the males (9.8 mmol/L, compared with the historical control of 3.0 - 5.6 mmol/L) and females (12.6 mmol/L, compared with the historical control of 3.92 - 8.2 mmol/L). In the mid-dose females and males, the urea level was significantly increased compared with the control groups. The protein level was decreased in high-dose males (60 g/L, compared with the historical control of 61 - 71 g/L) and females (57 g/L, compared with the historical control of 62 - 76 g/L). Mid-dose females also showed a significant decrease in protein levels. The urea and protein changes are probably related to the histopathological effects observed in the liver. These effects are considered to be adaptive, due to the increase metabolic load the test substance causes. The ALP level was increased in high-dose males (786 IU/L, compared with the historical control of 300 - 789 IU/L) and females (658 IU/L, compared with the historical control of 163 - 478 IU/L). The mid-dose females also had a significant increase in ALP levels. The ASAT level was increased in high-dose males (166 IU/L, compared with the historical control of 34 - 112 IU/L) and females (199 IU/L, compared with the historical control of 44 - 94 IU/L). The mid-dose females also had a non-significant increase in ASAT levels. Changes in hepatic enzyme levels indicate a higher metabolic load, considered to be adaptive effects related to the histopathological liver findings.

The cholesterol level was decreased in high-dose males (0.9 mmol/L, compared with the historical control of 1.2 - 2.8 mmol/L) and females (0.5 mmol/L, compared with the historical control of 1.1 - 2.7 mmol/L). In mid-dose females a significant decrease in cholesterol levels was observed. Furthermore, the triglyceride level was decreased in in high-dose males (0.13 mmol/L, compared with the historical control of 0.31 - 1.30 mmol/L) and females (0.18 mmol/L, compared with the historical control of 0.23 - 0.87 mmol/L). These effects may be related to the treatment with a fatty ester.

No organ weights were reported for the high-dose group, due to the early termination of the animals. A significant increase in relative liver weight in mid-dose males and females was noted, along with a significant increase in absolute liver weight in females of the same group. This is considered to an adaptive effect. The relative kidney weight was significantly increased in mid-dose males. As the increase was less than 10% and only observed in one sex, it is not considered to be toxicologically relevant. The absolute and relative ovary and testes weights were comparable between the control, low- and mid-dose groups.

Scabs were noted on the skin at the application site of 1/5 low-dose males and all the animals in the high-dose group. The liver showed an accentuated lobular pattern in 0/5, 0/5, 1/5 and 5/5 males in the control, low-, mid- and high-dose group, and in 0/5, 1/5, 4/5 and 5/5 females in the control, low-, mid- and high-dose group. In the high-dose group 3/5 males and 4/5 females had a pale liver, while in 1/5 high-dose males the liver also had a friable consistency. The liver effects are treatment-related, adaptive and consistent with the microscopic findings. The kidneys of 1/5 females in the mid-dose group, and of 3/5 males and of 1/5 females in the high-dose group, respectively, had a grey-green colour. The observation was correlated with the microscopic findings and considered to be related to the diet. In 2/5 high-dose males and 1/5 females the spleen was reduced in size, while the thymus was reduced in size in 1/5 high-dose females. The finding in the spleen and thymus are considered to be secondary to the general poor health of the animals.  

In all males and females of the high-dose groups ulceration, degeneration or necrosis and acanthosis of the skin was observed at the application site; caused by the treatment. 5/5 males and 5/5 females in the mid- and high-dose group, respectively, had hepatocellular hypertrophy. This effect is most likely an adaptive effect of the high hepatic load caused by exposure to the test substance (Bär, A. 1999). In the liver, minimal to marked steatosis was observed in 0/5, 0/5, 5/5 and 5/5 males in the control-, low-, mid- and high-dose group, and in 1/5, 5/5, 5/5 and 5/5 females in the control-, low-, mid- and high-dose group. The number of cases and the severity increased with increasing dose, indicating a treatment-related effect. Diets high in fatty acids are known to induce steatosis in rats and the effect is therefore considered to be an adaptive rather than an adverse effect (Buettner, R. et al. 2006).

Acidophilic globules in the cortical tubular epithelium of the kidney was observed in 5/5 male rats in all the treatment groups. The incidence and severity is considered to be caused by the increased production sex-specific alpha-2-microglobulin. This effect is only observed in male rodents and is not relevant to humans. The incidence of renal vacuolated cortical tubular epithelium in 1/5 females in the high-dose group is most likely a treatment-related effect (Altunkaynak, M.E. et al. 2008,). Cases of contracted spleen and lymphoid depletion of the thymus noted in 1/5 or 2/5 high-dose males and 1/5 high-dose females, respectively, is considered to be secondary to the poor general health condition of these animals as a consequence of the severe skin effects. As noted above for the individual parameters, the effects noted at the mid-dose level had low incidence, were related to the fatty diet or otherwise treatment-related and not toxicologically relevant. Based on the toxicologically relevant effects observed at the highest dose level, the NOAEL systemic and local is considered to be 300 mg/kg bw/day.

 

Overall conclusion for repeated dose toxicity

In the oral 28-day repeated dose toxicity study, adverse effect were observed at the high and medium dose levels of 300 and 1000 mg/kg bw/day, leading to a NOAEL systemic = 100 mg/kg bw/day. In the dermal 14-day repeated dose toxicity study severe local skin irritation /corrosion effects and were noted at the highest dose level of 860 mg/kg bw/day, no NOAEL could be derived.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The reliable GLP-compliant study performed similar to OECD guideline 407 was selected.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The reliable GLP-compliant study performed similar to OECD guideline 410 was selected.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
The reliable GLP-compliant study performed similar to OECD guideline 407 was selected.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Repeated dose toxicity: dermal - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

The available data on oral and dermal repeated dose toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.