3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate

Repeated dose toxicity: dermal

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate

EC Number:

261-665-2

EC Name:

3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate

Cas Number:

59219-71-5

Molecular formula:

C18H36O2

IUPAC Name:

3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate

Details on test material:

- Name of test material (as cited in study report): isononyl isononanoate
- Physical state: colourless liquid
- Analytical purity: no data
- Storage condition of test material: at room temperature
- Other: density 0.86

Test animals

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)

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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)

3

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

4

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)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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