Amides, tall-oil fatty, N,N-di-Me

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 March 2013 to 13 November 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliable, GLP and OECD Test Guideline No 422 compliant study providing results from a proprietary screening study.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amides, tall-oil fatty, N,N-di-Me

EC Number:

269-665-4

EC Name:

Amides, tall-oil fatty, N,N-di-Me

Cas Number:

68308-74-7

Molecular formula:

not applicable for UVCB

IUPAC Name:

Amides, tall-oil fatty, N,N-di-Me

Constituent 2

Reference substance name:

DMATO

IUPAC Name:

DMATO

Constituent 3

Reference substance name:

Dimethylamide van Talolie

IUPAC Name:

Dimethylamide van Talolie

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): DMATO
- Analytical purity: 100%
- Lot/batch No.: 12J46399
- Expiration date of the lot/batch: October 2015
- Storage condition of test material: At room temperature (20 ± 5°C) and protected from light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:male and female Wistar Hannover RccHan:WIST rats, obtained from Harlan Laboratories Models, S.L
- Age at study initiation: 11-12 weeks
- Weight at study initiation: Males: 346-383g; Females: 190-225g
- Fasting period before study: not applicable
- Housing:in standard laboraotory caging, group housd prior to mating; pair housed one male and one femal during mating phase and hosused individually subsequently
- Diet (e.g. ad libitum): Pelleted Harlan Teklad 2014C or 2018C rat/mouse maintenance diet provided ad libitum
- Water (e.g. ad libitum): Bottled tap water available ad libitum
- Acclimation period:The rats were acclimatised for at least 5 days prior to the start of the study.

Experimental dates 25 March 2013 to 13 November 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The amount of test material and administration volume was determined for each animal based on the most recent body weight. The dose volume was 4 mL/kg bw. No correction for purity was applied to the administered dose.

Dose levels were selected based on a dose range-finding study (Study S41514). Dose levels of 0, 50, 300 and 1000 mg/kg bw/day were chosen for the main study.
Oral gavage administration daily for two weeks prior to mating; males were dosed daily for at least 48 days after mating up to and including the day before sacrifice; the females were dosed up to and including the day before sacrifice (day 4 postpartum).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aliquots were taken from each of the dose formulations and analysed for concentration and homogeneity using GC-FID. Analyses were completed twice during treatment in the first week of treatment and during the post-pairing period. Homogeneity analyses were completed during the first week of treatment.
The results obtained showed the concentrations in the formulations ranged from 86.0% to 117.1% (within the accepted range of 80-120%) and the CV for homogeneity did not exceed 6%.

Duration of treatment / exposure:

Males: two weeks prior to mating and at least up to and including the day before sacrifice (at least a total of 28 days).
Females: two weeks prior to mating and at least up to and including the day before sacrifice (day 4 postpartum).

Frequency of treatment:

Daily.
Day 1: First treatment
Day 1 to 15: - pre-pairing treatment (checks for morbidity, bodyweight, food consumption and clinical signs)
Day 15: mating (checks for morbidity, bodyweight, clinical signs and evidence of mating - vaginal smears)
Day 16 - Day 0 of pregnancy
Day 16 to 37 - gestation (checks for morbidity, bodyweight, food consumption and clinical signs)
Day 37: Delivery
Day 38: Lactation F0 females (checks for nursing, morbidity, bodyweight, food consumption and clinical signs)
F1 litter size , sex, bodyweight, clinical signs and a behaviour test
F0 males (checks for morbidity, bodyweight and clinical signs)

Day 41: End of treatment (F0 females: day 4 postpartum (checks for grip strength, motor activity, sensory reactivity to stimuli) and F1 necropsy; F0 males (grip strength, motor activity, sensory reactivity to stimuli).

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 300, 1000 mg/kg bw/d
Basis:
other: nominal

No. of animals per sex per dose:

10 rats/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were chosen based on a 14-day range finding study

Positive control:

Not required ofr this study type

Examinations

Observations and examinations performed and frequency:

The animals were observed for morbidity/mortality at least twice daily. Cage-side clinical observations were made at least once daily.
Detailed clinical observations were performed on all test and control group animals before the first exposure, once weekly thereafter and one day before sacrifice. These observations were performed outside the home cage in a standard arena, at least one hours after dosing.
Functional performance tests (grip strength; fore and hind limbs and motor activity) were conducted in five randomly selected males per group once during the final week of treatment and at least one hour after dosing. Five randomly selected females per group were similarly evaluated on day 4 postpartum. Sensory reactivity to different stimuli (e.g. auditory, visual and proprioceptive) were evaluated in the same animals subject to functional performance tests.
Food consumption was determined once weekly in males during the pre-pairing period and two weeks of postpairing. Food consumption in females was determined once weekly during the pre-pairing period and days 0-7, 7-14, 14-21 post coitum and days 1-4 post partum. Body weights were recorded for males twice weekly during the pre-pairing and pairing period and daily during the postpairing period. Female body weights were recorded twice weekly during the pre-pairing and pairing period and daily until day 4-5 post partum. Blood was collected from 5 males on at least day 43 of treatment (day of sacrifice) and from 5 females on approximately day 5 post partum (day of sacrifice) for clinical laboratory investigations.

From day 21 postcoitum, the females were examined at least twice daily for signs of parturition. Gestation length was recorded.
For the F0 generation twice daily checks were made for morbidity and mortality and once daily cage-side checks of clinical signs. A detailed behavioural assessment was completed for the test and control animals at weekly intervals. A functional performance test for grip strength and motor activity, sensory reactivity assesssments for auditory, visual and proprioceptive stimuli were evaluated. Food consumption was measured for the males at weekly intervals during the pre-pairing period and during two weeks of the post-mating phase and at weekly intervals through the study for the females.
water consumption was recorded for males during day 7-15 post-pairing and for females on days 6-14 post coitum.
Bodyweights were recorded for at weekly intervals pre-pairing and daily thereafter.
Vaginal smears were collected from all females and examined for oestrous cycling during the mating phase. Smears were discontinued when sperm were found.
The females were checked from day 21 postcoitum for signs of parturition and gestation length was recorded. The females that gave birth were observed to determine if they nursed their young.

Sacrifice and pathology:

Females were sacrificed on approximately day 5 post partum and necropsied. Males were sacrificed on at least day 43 of the study. All sacrificed animals were examined macropscopically. Tissues were collected for histopathological processing and determination or organ weights.
Any rat sacrificed or found dead during the study was examined macroscopically. Gross macroscopic examination was performed of all internal organs with emphasis on the uterus, count of corpora lutea and implantation sites. If no implantation sites were found then the uterus was placed in an aqueous solution of ammonium sulphide to highlight implantation sites. F0 females were terminated on day 5 post partum. The mated females that did not give birth or show any signs of pregnancy (five individuals numbers 58, 62, 65, 71 and 78) were terminated 26 days post coitum.

Other examinations:

A qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high dose groups was performed.

Statistics:

Dunnett's test, Fisher's Exact test and Steel's test were variously used on study parameters

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was recorded in males and females from control and at 50, 300 and 1000 mg/kg bw/day, with greater incidence at 1000 mg/kg bw/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

Salivation was recorded in males and females from control and at 50, 300 and 1000 mg/kg bw/day, with greater incidence at 1000 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower body-weight gain was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day during the premating period;

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Higher water consumption was recorded in both sexes at 1000 mg/kg bw/day.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

An increase in reticulocyte values and white blood cells was observed in males at 1000 mg/kg bw/day

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

An increase in cholesterol and triglyceride levels was recorded at 300 and 1000 mg/kg bw/day in males and at 1000 mg/kg bw/day in females. Moreover, increases in alkaline phosphatase and alanine aminotransferase were recorded at 1000 mg/kg bw/day

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Lower locomotor activity was recorded in both sexes at 300 and 1000 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

An increase in liver weight was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day. Moreover, higher kidney weight was recorded in males at 1000 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Reddish discoloration of the mesenteric lymph node was observed in 0/10, 0/10, 0/10 and 5/10 males and in 0/10, 0/10, 1/10 and 2/10 females at 0, 50, 300 and 1000 mg/kg bw/day, respectively.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

various findings in both sexes at 1000 mg/kg bw/day. Hepatocellular hypertrophy was seen in males at 300 mg/kg bw/d

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY

No deaths occurred.

CLINICAL SIGNS

Salivation was recorded in males and females from control and at 50, 300 and 1000 mg/kg bw/day, with greater incidence at 1000 mg/kg bw/day.

BODY WEIGHT AND WEIGHT GAIN

Lower bodyweight gain was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day during the premating period; afterwards there was a recovery in bodyweight. No test-item related differences from the control group were recorded at 50 mg/kg bw/day.

FOOD CONSUMPTION

There were no effects of treatment

WATER CONSUMPTION

Higher water consumption was recorded in both sexes at 1000 mg/kg bw/day. No significant differences were recorded at 50 and 300 mg/kg bw/day.

HAEMATOLOGY

In males at 300 and 1000 mg/kg bw/day, reticulocyte values, median fluorescence reticulocyte ratio (MFR) and low fluorescence reticulocyte ratio (LFR) increased with respect to the control group. These differences were statistically significant at 1000 mg/kg bw/day and, only in reticulocyte count, at 300 mg/kg bw/day. In addition, higher white blood cell values were recorded at 1000 mg/kg bw/day. No differences with the control groups were recorded at 50 mg/kg bw/day or in females of any group.


CLINICAL CHEMISTRY

An increase in cholesterol and triglyceride levels was recorded at 300 and 1000 mg/kg bw/day in males and at 1000 mg/kg bw/day in females. Moreover, increases in alkaline phosphatase and alanine aminotransferase were recorded at 1000 mg/kg bw/day in males and females. No relevant differences were observed at 50 mg/kg bw/day.

NEUROBEHAVIOUR

Lower locomotor activity was recorded in both sexes at 300 and 1000 mg/kg bw/d and in males at 50 mg/kg bw/day. No relevant differences from the control group were recorded in the grip strength test in either sex. All animals showed a normal behavior and appearance. All animals responded positively to the different reflexes such as palpebral, righting and iridial reflexes and pain, auditory startle and handling response (push-off).

ORGAN WEIGHTS

Higher liver weights were recorded at 300 and 1000 mg/kg bw/day in males and at 1000 mg/kg bw/day in females. These differences were statistically significant in relative to body and brain weight in both sexes at 1000 mg/kg bw/day. In addition, a significantly higher kidney weight was recorded at 1000 mg/kg bw/day in males. A higher thymus weight at 300 and 1000 mg/kg bw/day and adrenal weight at 1000 mg/kg bw/day was observed in females. These differences were not statistically significant. No relevant differences from the control group were recorded at 50 mg/kg bw/day.
Concerning reproductive organs, lower weight of the testes was recorded in males no. 18 at 50 mg/kg bw/day, 29 at 300 mg/kg bw/day and 33 at 1000 mg/kg bw/day. No differences in weight were recorded in female reproductive organs.


GROSS PATHOLOGY

No treatment-related alterations were recorded in the offspring at necropsy. Reddish discoloration of the mesenteric lymph node was observed in 0/10, 0/10, 0/10 and 5/10 males and in 0/10, 0/10, 1/10 and 2/10 females at 0, 50, 300 and 1000 mg/kg bw/day, respectively. All other lesions recorded were considered to be within the normal range of background alterations observed in rats of this strain and age and under the experimental conditions used in this study.

HISTOPATHOLOGY

At 1000 mg/kg bw/day, slight hepatocellular hypertrophy in the liver was recorded in males and females, the enlarged hepatocytes displaying a “ground glass appearance”. In the thyroid minimal to moderate follicular cell hypertrophy was observed in males and females; the changes were characteried by the presence of irregularly shaped follicles lined by columnar cells and containing a small amount of pale-staining colloid. In the pituitary, minimal cell hypertrophy was recorded in the pars distalis of 1/10 males; the finding was characterized by an increased number of scattered enlarged pale eosinophilic cells. Moreover, a decrease in lymphocytes was observed with higher incidence in the mesenteric lymph node of males and females and in the thymus of males. In kidneys, hyaline droplets within the proximal tubule lining cells were recorded with higher incidence and severity in males compared to the control group. Non-pregnant rat no. 71 had diffuse vacuolation of the ovaries (involving interstitial, luteal and follicular cells) and vacuolation of the epithelial cells lining the oviducts. In this animal, vacuolation was also present in the enlarged adrenals observed at necropsy. In the other females, the ovaries, oviducts, uterus, cervix and vagina were unremarkable, in particular the ovarian follicles and corpora lutea. At 300 mg/kg bw/day, minimal hepatocellular hypertrophy in the liver was recorded in males., but not in females. At 50 mg/kg bw/day, No hepatocellular hypertrophy in the liver was recorded in males or in females. All other findings recorded were within the range of normal background lesions which may be
recorded in animals of this strain and age.

OTHER FINDINGS

Sperm Stage Evaluation revealed severe diffuse bilateral tubular degeneration and was associated with marked oligospermia or aspermia in the epididymides was recorded in males that showed small testes and/or epididymides. No remarkable findings were recorded in the remaining males from highand control groups.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: The NOAEL for males of 50 mg/kg bw/day is based on increased liver weight, hepatocyte hypertrophy and a significant increase in serum cholesterol at 300 mg/kg bw/d.

2

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: The NOAEL for females of 300 mg/kg bw/d is based on reduced weight gain, increased liver weight, hepatocyte hypertrophy and a significant increase in serum cholesterol at the highest dose level of 1000 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Summary of in-life findings

Parameter	M				F
	0	50	300	1000	0	50	300	1000
Locomotor activity (counts)	1563	1097*	764**	647**	823	767	677	597
Water consumption (g/day)	26.8	29.9	30.5	44.8	27.3	35.6	32.9	45.6
Pre-mating weight (g)	410.4	410.2	397.5	384.2**	226.9	225.1	222.7	226.6
Terminal weight (g)	454.3	457.3	437.2	422.4**
Reticulocytes (%)	1.4	1.7	3.4*	2.0*	5.0	7.7	5.3	5.9
Reticulocytes (G/L)	132.2	157.5	283.6	184.3	360.6	523.4	376.3	441.3
LFR	0.776	0.726	0.654	0.698*	0.478	0.478	0.505	0.543
MFR	0.213	0.258	0.302	0.282*	0.417	0.420	0.394	0.392
HFR	0.011	0.016	0.044*	0.019	0.105	0.102	0.101	0.065
WBC (G/L)	6.6	7.6	7.3	8.8**	5.9	6.3	5.7	5.6
Lymphocytes (G/L)	5.1	6.1	5.3	6.9*	3.7	3.7	3.3	3.5
Cholesterol (mM)	2.01	1.80	2.65*	3.16**	1.77	2.56	2.43	2.89*
ALT (U/L)	33.3	30.8	33.3	45.4*	35.6	39.7	44.4	45.0
ALP (U/L)	61.8	49.5	49.8	90.8*	36.1	34.4	36.8	47.5
P (mM)	1.69	1.71	1.88	1.95*	2.23	2.35	2.16	2.26
Na (mM)	144.3	144.2	145.6	146.7**	138.9	139.9	139.0	139.1
Cl (mM)	99.8	99.1	100.1	103.1**	100.1	100.9	100.5	100.5
Protein (g/L)	67	68	68	63*	62	65	67	65
Globulin (g/L)	17	20	20	22*	23	23	22	19
A:G ratio	2.59	2.31	2.28	1.94*	1.96	1.92	2.11	2.29

*significantly different to controls (p<0.05); **p<0.01

Summary of organ weight effects

Organ	M				F
	0	50	300	1000	0	50	300	1000
Liver weight (g)	10.38	10.80	11.35	14.18**	8.42	8.62	8.66	10.82**
Liver weight (%)	2.35	2.45	2.73**	3.55**	3.44	3.48	3.45	4.41**
Liver weight (% brain)	491.4	522.3	540.1	676.3**	453.2	468.6	466.5	583.2**
Thymus weight (g)	0.361	0.400	0.301	0.288	0.190	0.195	0.237	0.234
Thymus weight (%)	0.082	0.091	0.072	0.072	0.076	0.079	0.094	0.096
Thymus weight (% brain)	17.0	19.4	14.3	13.7	10.2	10.6	13.0	12.7
Kidney weight (g)	2.31	2.41	2.40	2.56*	1.52	1.50	1.56	1.63
Kidney weight (%)	0.52	0.55	0.58*	0.64**	0.62	0.61	0.62	0.66
Kidney weight (% brain)	109.0	116.3	114.2	121.9**	82.0	81.6	84.5	87.7
Prostate weight (g)	2.95	3.01	2.78	2.52*
Prostate weight (%)	0.67	0.68	0.67	0.63
Prostate weight (% brain)	139.6	15.8	132.4	120.0

*significantly different to controls (p<0.05); **p<0.01

Summary of necropsy findings

Finding	M				F
	0	50	300	1000	0	50	300	1000
Mesenteric lymph node: discoloured	-	-	-	5	-	-	1	2
Mesenteric lymph node: erythrocytosis	-	-	-	5	-	-	1	2
Hepatocyte hypertrophy	-	-	4	9	-	-	-	5
Kidney hyaline deposition	-	-	-	5	-	-	-	-

Applicant's summary and conclusion

Conclusions:

F0 generation - Males
Based on the results obtained, the dose of 50 mg/kg bw/day can be considered the NOEL (No Observed Effect Level) for toxic effects, and 300 mg/kg bw/day, the LOAEL (Low Observed Adverse effect Level) based on an increase in hepatocellular hypertrophy in liver and hyaline droplets within tubular epithelium in kidneys at 300 and 1000 mg/kg bw/day.

The dose of 1000 mg/kg bw/day can be considered the NOAEL (No Observed Adverse Effect Level) for fertility and mating performance.
F0 generation - Females
Based on the results obtained, the dose of 300 mg/kg bw/day can be considered the NOAEL for toxic effects, and 1000 mg/kg bw/day, the LOAEL based on an increase in hepatocellular hypertrophy in liver at 1000 mg/kg bw/day. The dose of 50 mg/kg bw/day can be considered the NOEL and 300 mg/kg bw/day, the LOAEL for fertility and mating performance based on the lower implantation sites and corpora lutea recorded at 300 and 1000 mg/kg bw/day.

F1 generation
The dose of 1000 mg/kg bw/day can be considered the NOAEL.

Executive summary:

 This study was performed to OECD Guideline 422 [Combined repeated Dose Toxicity Study with the Reproduction /Developmental Toxicity Screening Test] with the Submission Substance DMATO. Groups of RccHan:WIST rats were gavaged with DMATO (in corn oil) at dose levels of 0 (vehicle control), 50, 300 or 1000 mg/kg bw/d at a constant dose volume of 4 mL/kg bw/d. Animals were dosed for a two week pre-mating period, throughout mating, and during pregnancy and early lactation for females). The dosing period for males was at least 49 days.

Clinical signs, behavioural assessments, body weights and food and water consumption were monitored during the study. Females were also frequently examined on days 21 and 22 of pregnancy in order to check delivery. Haematology and clinical biochemistry were evaluated at termination on five selected males and females from each dose group. Pairing of animals within each dose group was undertaken on a 1:1 basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring up to Day 4 of lactation. During the lactation phase, clinical observations were recorded daily for all surviving offspring, together with litter size and offspring weights; and surface righting reflex was assessed. Extensive functional observations were performed on five selected males from each dose group the day before sacrifice and for five selected parental females from each dose group on Day 4 post partum. Adult males were terminated at least on Day 49, adult females on Day 5 post partum and offspring on Day 4 post partum. All animals were subjected to a gross necropsy examination and selected tissues from five males and five females from groups 1 and 4 were evaluated histopathologically.

No mortality was recorded during the study period. Salivation was recorded in males and females in all groups, with a greater incidence at 1000 mg/kg bw/day. Lower locomotor activity was recorded in both sexes at 300 and 1000 mg/kg bw/day. No relevant differences from the control group were recorded in the grip strength test in either sex. Sensory reactivity assessment did not reveal any differences from the control group; all animals showed a normal behaviour and appearance.

Higher water consumption was recorded in both sexes at 1000 mg/kg bw/day; no differences were recorded at 50 and 300 mg/kg bw/day. No relevant differences from the control group were recorded in food consumption. Lower body-weight gain was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day during the pre-mating period; afterwards there was a recovery in bodyweight. No test-item related differences from the control group were recorded at 50 mg/kg bw/day.

Haematology revealed an increase in reticulocyte values and white blood cells males at 1000 mg/kg bw/day; no relevant differences were observed in the remaining groups or females. Clinical chemistry assessment showed an increase in cholesterol and triglyceride levels at 300 and 1000 mg/kg bw/day in males and at 1000 mg/kg bw/day in females. Increases in alkaline phosphatase and alanine aminotransferase were recorded at 1000 mg/kg bw/day in males and females.

Gross necropsy revealed a reddish discoloration of the mesenteric lymph node in both sexes at 1000 mg/kg bw/day. All other gross lesions recorded were considered to be within the normal range of background alterations observed in rats of this strain and age and under the experimental conditions used in this study.

An increase in liver weight was recorded in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day. Moreover, higher kidney weight was recorded in males at 1000 mg/kg bw/day. One male from each test-item-treated group had lower testis weight. No noticeable differences were observed in the remaining animals.

 

At 1000 mg/kg bw/day, cell hypertrophy in the liver, thyroid and pituitary consistent with liver enzyme induction, as well as, decreased lymphocytes in the mesenteric lymph node and thymus were recorded in both sexes. Increased hyaline droplets in the kidneys were observed in males. The hyaline droplets observed in high dose males are consistent with species specific male kidney nephropathy arising from administration of high oil content doses that may exacerbate the accumulation of alpha2μ-globulin, a unique protein occurring spontaneously in proximal convoluted tubular epithelial cells only in the mature male rat. Thus, the male rat hydrocarbon nephropathy should not be predictive of a normal human renal response. One female at 1000 mg/kg bw/day had vacuolar changes (involving ovary, oviduct and adrenals). At 300 mg/kg bw/day, cell hypertrophy in the liver was recorded in males, but not in females.

Sperm Stage Evaluation showed severe diffuse bilateral tubular degeneration, associated with marked oligospermia or aspermia in the epididymides in males that showed small testes and/or epididymides. No remarkable findings were recorded in the remaining males from high and control groups.

A NOAEL for males of 50 mg/kg bw/day is determined, based on increased liver weight, hepatocyte hypertrophy and a significant increase in serum cholesterol at 300 mg/kg bw/d. A NOAEL for females of 300 mg/kg bw/d is based on reduced weight gain, increased liver weight, hepatocyte hypertrophy and a significant increase in serum cholesterol at the highest dose level of 1000 mg/kg bw/d.