Naphthenic acids, bismuth salts

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: combined repeated dose & reproductive/developmental toxicity screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is performed by NTP according to GLP and valid methods, therefore it is considered to be adequate, reliable and relevant for classification. The score 1 was given by HPVIS.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is performed by NTP according to GLP and valid methods, therefore it is considered to be adequate, reliable and relevant for classification. The score 1 was given by HPVIS.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, 2000/OECD 422

Deviations:

no

Principles of method if other than guideline:

see section 7.5.1. (Repeated dose toxicity), section 7.6.2. (Genetic toxicity in vivo), section 7.8.1. (Toxicity to reproduction)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at study initiation: no data
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: To: no data

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The naphthenic acids were suspended in corn oil to the appropriate concentrations and administered in 10 ml/kg doses.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days. All females confirmed to have mated were placed in plastic maternity cages once mating was confirmed.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing females were plac- Further matings after two unsuccessful attempts: Females for which copulation was not detected were placed in maternity cages at the end of the 14 day mating period.
- After successful mating each pregnant female was placed in maternity cages once mating was confirmed
- Any other deviations from standard protocol: Length of gestation was calculated as the time from confirmation of mating to the onset of delivery.

Duration of treatment / exposure:

Dosing initiated 14 days prior to pairing and continued throughout the mating and gestational periods until study termination on post-natal day 3. The total number of doses ranged from 39 - 53 depending on the time at which mating occurred.

Frequency of treatment:

Daily

Duration of test:

39 - 53 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- All rats were examined twice daily for mortality and general health.
- All animals were examined approximately 1 hour after each treatment, and all unusual observations were recorded. ed.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Detailed physical examinations of all animals were conducted weekly (See Section 7.5.1)

BODY WEIGHT: Yes
- Time schedule for examinations:females: recorded once week prior to test substance administration, on the first day of dose administration and weekly until evidence of copulation was obtained. From that point body weights of female rats were recorded on gestation days (GD) 0, 4, 7, 11, 14, 17, and 20 and on lactation days (LD) 0, 1 and 4 (termination). For females for which there was no evidence of copulation, body weights were recorded weekly until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption by adult animals was also recorded on the same schedule as the body weights.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #4, Females for which there was no evidence of mating were sacrificed on post-cohabitation day 25, those that showed evidence of mating but failed to deliver were euthanized on post-mating day 25, and all others were euthanized on post-natal day 4
- An examination of target organs including male and female reproductive organs was also carried out as part of this test (See also Section 7.5.1).
- Organs examined included: ovaries with oviduct, uterus with cervix and vagina, testes with epididymides, prostate and seminal vesicles.
- The ovaries, testes and uteri were weighed and all were examined histologically

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other: Uteri with no microscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

Parental mating, fertility, conception and copulation indices were analyzed using the Chi-square test with Yates’ correction (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites, numbers of corpora lutea, number of pups born, live litter size on PND 0, unaccounted for sites, and pre-coital intervals were evaluated by one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences between the vehicle control and test substance-treated groups. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent of litter) of males at birth and post-natal survival were evaluated using the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences between the vehicle control and test substance-treated groups. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunn Test (Dunn, 1964) was used to compare the test substance-treated groups to the vehicle control group.

Indices:

See Tables below and in Section 7.8.2.
Mating, fertility, pregnancy and gestation indices were not provided as such, however No. of females mated, pregnant and with litters were given.
Pre-implantation loss not provided, but No. of corpora lutea and No. of implantation sites given.
Implanation index not provided, but No. of implanatation given.
Post-implantation indexes given.

Details on maternal toxic effects:

Maternal toxic effects: no effects

Details on maternal toxic effects:
Althouth it was mentioned above that NOAEL for maternal toxicity is 900 mg/kg bw, the toxicity part of this experiment learned that systemic toxicity and target organs were identified at 300 and 900 mg/kg bw. Therefore, it can be considered that there was maternal toxicity.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

ca. 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

There were no apparent effects on mating. A single female in the 300 mg/kg/day group had a pre-coital interval of 13 days, resulting in a statistically significant increase in pre-coital incidence in this group. Otherwise all of the pairs productively mated and pre-coital intervals were within the historical control range for the laboratory. Note that there was a significant increase in pre-coital interval in the 300 mg/kg group, but this was due to a single female, and, as noted was within the historical range of the laboratory. Accordingly, it was not considered to have been a treatment-related effect. 

The length of the gestational period was similar across the groups. There were reductions in the numbers of corpora lutea and implantation sites in the high dose group, but the differences were not statistically significant (see Table 1 below). However, there was a significant reduction in the number of offspring born/litter in the high dose group (Table 2). There was also a significant reduction in survival in offspring in the high dose group, and those that did survive had significantly lower body weights than the offspring in the control groups. The number of pups found dead or euthanized in extremis during the period PND 0 - 4 was: control = 1(1), 100 mg/kg/day = 0(0), 300 mg/kg/day = 12(5), and 900 mg/kg/day = 38(8).  

Table 1. Summary of reproductive parameters assessed in the repeated dose/reproductive toxicity study of refined naphthenic acids

Dose (mg/kg/day)	Corn Oil Control	100 mg/kg/day	300 mg/kg/day	900 mg/kg/day
Number of females paired	12	12	12	12
Number of female mated	12	12	10	11
Number of females pregnant^a	9	12	10	11
Number of females with litters	9	12	10	11
Pre-coital interval (days)^b	1.4+0.7	2.3+1.1	4.2+3.3*	3.8+3.5
Gestation length (days)	21.4+0.6	21.9+0.3	22.0+0.5	22.1+0.5
Corpora lutea	15.6+2.3	14.0+1.4	15.1+3.0	13.8+2.1
Implantation sites	15.0+2.4	13.6+1.1	13.0+1.2	12.2+3.7
Number born	14.1+1.9	12.9+1.1	12.0+1.6	10.8+3.8^c
Post-Implantation loss (%)^d	6.0	5.1	7.7	11.5

a. Pregnant = uterine implantation sites.

b. Data summarized as mean+standard deviation.

c. p < 0.05

d. Post-implantatoin loss = (No. of implantations - No. of life fetuses)/No. of implanatations (%)

 

Table 2. Survival, viability and growth of offspring following in utero exposure to refined naphthenic acids. The data are given as mean+SD.

Dose (mg/kg/day)	Corn Oil	100 mg/kg/day	300 mg/kg/day	900 mg/kg/day
Number of viable litters	9	12	10	11
Number of pups born alive/litter	13.9+1.9	12.9+1.1	10.1+4.0^a	9.6+4.0^b
Percentage of pups surviving from birth to termination        ( PND 4)	98.1+3.8	100.0+0.0	88.0+24.5	67.7+40.6
Pups (litters) found dead or euthanizedin extremis	1(1)	0(0)	12(5)	38(8)
Sex ratio (% males/litter)	58.9+9.6	53.9+9.6	55.2+19.1	58.1+22.7
Pup weight PND 1 – males	7.0+0.5	6.7+0.7	6.7+0.5	5.7+0.8^a
Pup weight PND 1 – females	6.6+0.6	6.5+0.6	6.4+0.4	5.6+1.1
Pup weight PND 4 – males	9.7+1.1	9.4+1.2	9.4+0.9	7.2+1.5^b
Pup weight PND 4 – females	9.1+1.0	9.0+1.0	8.8+0.7	7.3+1.5^b

a. p < 0.05, b. p < 0.01

Conclusions:

Treatment of Sprague-Dawley rats with refined naphthenic acids had no apparent effects on mating and did not produce malformations at the highest dose tested (900 mg/kg/day). However, there were significant reductions in number of offspring, number live born and offspring body weights. The overall no observed adverse effect level was 100 mg/kg/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats was performed by oral gavage with naphtenic acids in corn oil. There were 3 test material treated groups (100, 300 and 900 mg/kg bw) along with a vehicle treated group (corn oil) each in 12 animals/sex/group. Male rats were dosed during premating, mating and afterwards for 28 days in total and females were dosed during premating, mating, gestation and up to day 3 post partum. In this section, only developmental toxicity parameters are discussed: (furher info on repeated & reproductive parameters is given in Section 7.5.1 and 7.8.1). Target organ findings were identified at the dose of 300 and 900 mg/kg bw, whereas 100 mg/kg bw was considered as the NOAEL for systemic toxicity.

Treatment of Sprague-Dawley rats with refined naphthenic acids had no apparent effects on mating and did not produce malformations at the highest dose tested (900 mg/kg/day). However, there were significant reductions in number of offspring, number live born and offspring body weights. The overall no observed adverse effect level was 100 mg/kg/day.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is performed by NTP according to GLP and valid methods, therefore it is considered to be adequate, reliable and relevant for classification. The score 1 was given bij HPVIS.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The study protocol has been amended by an in vivo study for mutagenicity in consultation with US EPA

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650

Deviations:

yes

Remarks:

The study protocol has been amended by an in vivo study for mutagenicity in consultation with US EPA

Principles of method if other than guideline:

An extension for in vivo micronucleus study was also integrated in the study (see 7.6.2.)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

no data

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The naphthenic acids were suspended in corn oil to the appropriate concentrations and administered in 10 ml/kg doses.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Males: dosing for 28 - 29 days
Females: Depending on the time at which mating occurred, females were dosed for 39 - 53 days

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- All rats were examined twice daily for mortality and general health.
- All animals were examined approximately 1 hour after each treatment, and all unusual observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Detailed physical examinations of all animals were conducted weekly.

BODY WEIGHT: Yes
- Body weights of male rats were recorded one week prior to test substance administration, on the first day of dose administration, on a weekly basis during the study and at termination. Body weights of female rats were recorded once week prior to test substance administration, on the first day of dose administration and weekly until evidence of copulation was obtained. From that point body weights of female rats were recorded on gestation days (GD) 0, 4, 7, 11, 14, 17, and 20 and on lactation days (LD) 0, 1 and 4 (termination). For females for which there was no evidence of copulation, body weights were recorded weekly until termination. Body weights of offspring were recorded on post-natal day (PND) 1 and then on PND 4, prior to termination.

FOOD CONSUMPTION:
- Food consumption determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No (gavage)

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No data
- On the day of scheduled termination
- The hematological investigation included measurements of total leukocyte count, erythrocyte count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, prothrombin time, activated partial prothrombin time, reticulocyte count, mean platelet volume, red cell distribution width, hemoglobin distribution width, differential leukocyte count, and red cell morphology.
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- On the day of scheduled termination
- The serum chemistry investigation included measurements of concentrations of albumin, total protein, globulin, albumin/globulin ratio, total bilirubin, urea nitrogen, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminostransferase, gamma glutamyltransferase, glucose, total cholesterol, calcium, chloride, phosphorus, potassium, sodium, triglycerides, and bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- The potential for nervous system effects was assessed using a functional observation battery (FOB). All rats in the vehicle (corn oil) and naphthenic acid-treated groups were examined prior to dosing, after approximately 28 days of dosing, and, for females, prior to termination. The FOB procedures were based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988; 1991; O’Donoghue, 1989). The testing was conducted in a sound attenuated room with a white noise generator set to operate at 70 + 10 dB. The investigators conducting the FOB were not aware of the treatment groups from which the respective animals were taken. The FOB consisted of the following: home cage observations; handling observations; open field observations; sensory observations and neuromuscular observations (Table 2). In addition there were physiological observations including body weight, body temperature and examination for catalepsy. There was also an assessment of locomotor activity which was measured electronically using a computer-controlled system with a series of infrared photobeams in a clear plastic rectangular cage. Animals were tested separately in 60 minute sessions divided into 5 minute intervals.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- At termination rats were euthanized by carbon dioxide inhalation.
- Necropsies were conducted on all animals sacrificed in extremis or at study termination.

ORGAN WEIGHTS
- Organs were removed weighed
- See table 1 and 3

HISTOPATHOLOGY: Yes
- Organs were placed in 10% neutral buffered formalin for histologic examination.
- The disposition of organs and tissues was as listed in the table 4.

Statistics:

Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption, body weight changes, absolute and relative organ weights, clinical pathology values (except for gamma glutamyltransferase), and continuous FOB values were evaluated by one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences between the vehicle control and test substance-treated groups. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Histopathological findings in the test substance-treated groups and FOB parameters yielding scalar or descriptive data were compared to the vehicle control group using Fisher’s Exact Test (Steel and Torrie, 1980). Gamma glutamyltransferase data were evaluated using the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences between the vehicle control and test substance-treated groups. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunn Test (Dunn, 1964) was used to compare the test substance-treated groups to the vehicle control group.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

2 mortalities and clinical observations seen in high dose females; clinical observations in some high dose group males.

Mortality:

mortality observed, treatment-related

Description (incidence):

2 mortalities and clinical observations seen in high dose females; clinical observations in some high dose group males.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain at high dose was reduced for less than 10% in males and 4% in females versus control groups (not statistically significant).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced in the high dosed male and female groups (statistically significant).

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):

Slightly reduced hemoglobin in males (statistically significant), however not in females.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Small incidental findings only, within historical range.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased liver weight in males and females of medium and high dose groups (only statistically significant at high dose). Significant increase in kidney weight in males of medium and hig dose group (only statistically significant at high dose).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pale kidneys in the high dose males and reduction in the number of corpora lutea in the high dose females

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hyaline-droplets in high dosed male kidneys (not relevant for humans); liver hepatocellular hypertrophy in high dosed males & females (adaptive); thyroid gland hypertrophy and vacuolation in (mid) and high dosed males & females (compensatory).

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Two high dose females were terminated on Lactation Day 2; one was sacrificed in extremis due to acute inflammation of the uterus; the other was sacrificed due to total litter loss. All other rats survived to scheduled termination. Clinical observations, which were noted only in high dose group females and approximately an hour of dosing included hunched posture; rocking, lurching, and/or swaying while ambulating; walking on tiptoes; hypoactivity; and shallow respiration. Some of the high dose group males also exhibited hunched posture.
BODY WEIGHT AND WEIGHT GAIN, FOOD CONSUMPTION
Body weight gain was reduced in high dose group males but the overall difference was less than 10% and the differences were not statistically significant. Among the females, the body weight gain in the high dose group was approximately 4% below control values but not significantly different at the end of the mating period. These differences in weight gain were associated with significantly reduced food consumption in the high dose group animals.
HAEMATOLOGY
There were some haematology changes, primarily reductions in parameters related to haemoglobin content which were considered to have been treatment related. However, as is apparent from Table 1, the differences were small and there was no consistency between males and females.
CLINICAL CHEMISTRY
The clinical chemistry values showed a similar pattern. Among males the only statistically significant differences between control were for creatinine (control value = 0.3 ±0.1 mg/dL versus a value of 0.4 ±0.0 in the high dose group, p < 0.01), and chloride (control value = 104 ±1.1 mEq/L versus a value of 102 ±1.3 in the high dose group, p < 0.01). Among the female rats, statistically significant differences were found for albumin (control = 4.3 ±0.2 g/dL versus 4.7 ±0.3 in the high dose group, p < 0.05), total protein (control = 6.3 ±0.3 g/dL versus 6.7 ±0.4 in the high dose group, p < 0.05), glucose (control = 115 ±11 mg/dL versus 130 ±8.0 in the high dose group, p < 0.05), cholesterol (control = 69 ±14 mg/dL in the control versus 89 ±19 in the high dose group, p < 0.05), calcium (control = 10.6 ±0.4 mg/dL in the control versus 11.5 ±0.6 in the high dose group, p < 0.01), and phosphorus (control = 3.9 ±0.6 mg/dL versus 5.5 ±1.2 in the high dose group, p < 0.05). All of the differences were small and within the historical range of the laboratory. Additionally, most were significant at only the 0.05 level, and there was no consistency of response between the sexes. In the absence of any corresponding pathological findings, these differences were most likely incidental.
NEUROBEHAVIOUR
There were no statistically significant differences in parameters assessed as part of the functional observation battery including home cage observations, handling parameters, open field observations, sensory observations or neuromuscular observations. There were some small differences in body weight gain as indicated previously but other physiological parameters (catalepsy, body temperature) were not affected by treatment. There were also no differences in locomotor activity patterns.
ORGAN WEIGHTS
Organ weight determinations in males revealed significant increases in weights of liver, kidney, thyroid/parathyroid and epididymis although the differences in thyroid/parathyroid and epididymal weights were only statistically different when compared on a relative to body weight basis. In females, there was a significant increase in liver weights and significant reductions in lung weights, and absolute uterine weights (table 3). The lung weights were within the historical range for the laboratory and were not associated with any pathological changes. The uterine weights were not significantly different when compared relative to body weights. All gravid females were in lactational anaestrus and undergoing involution. Uterine weight values all fell within the historical range for the laboratory and were not associated with any gross, histopathologic or clinical pathology changes. Other than the uterine weights there were no microscopic differences in the reproductive organs of the male and female rats.
GROSS PATHOLOGY
The only notable gross observations were those of pale kidneys in the high dose males and a reduction in the number of corpora lutea in the high dose group females. Otherwise, the results of the gross examination were not remarkable.
HISTOPATHOLOGY: NON-NEOPLASTIC
The results of the pathological investigation are summarized in Table 4. Kidney changes, reported in male rats only, were consistent with hyaline-droplet nephropathy (α2u-globulin-mediated nephropathy). The liver changes, found in organs from both male and female rats from the high dose group, were described as hepatocellular hypertrophy. Other changes included cortical lymphoid depletion of the thymus in females, primarily in rats from the high dose group. Epithelial hypertrophy and cytoplasmic vacuolation of the thyroid gland was noted in all treated animals, and cytoplasmic vacuolation of the zona fasciculate in the adrenal cortex was reported in males from all treatment groups and in high dose group females. The microscopic examination also revealed minimal cardiomyopathy which occurred with increased incidence in the males in the 100, 300 and 900 mg/kg/day groups. The pathologist noted that cardiomyopathy is a common finding in rats (Greaves, 2007a), that the incidence of cardiomyopathy in the treated animals was within the historical range of the laboratory, and that the severity of cardiomyopathy in the treated male rats was similar to or less than the degree of severity found in the control animals. The pathologist also noted that the cardiomyopathy was not associated with any gross observations, organ weight changes or alterations in clinical pathology parameters.
HISTOPATHOLOGY: NEOPLASTIC
No neoplastic changes.
HISTORICAL CONTROL DATA
Not provided
OTHER FINDINGS
The gross and pathological assessments did reveal some differences that were treatment-related but were unlikely to have been toxicologically important. Liver weights were significantly increased in high dose groups of both male and female rats, and there was also a statistically significant increase in liver weight in the 300 mg/kg/day dose group in the males. The histological findings were essentially limited to minimal evidence of hepatocellular hypertrophy in the high dose group animals. As none of the liver enzyme markers were increased, this was most likely evidence of enhanced metabolic capacity and adaptive rather than adverse (Cattley and Popp, 2002). Kidney weights were significantly elevated in the male rats from the high dose group, but not in the female rats. The histological evidence revealed the presence of hyaline droplets, mostly judged to have been of minimal severity, which increased in frequency in the male rats in a dose-dependent manner. As these were not found in female rats, the histological findings and gender-specificity, suggest the kidney changes were the consequence of an α-2u-globulin-related process which is male rat specific and not relevant to humans (Hard et al., 2008; Baetcke et al., 1991; Swenberg and McKeeman, 1998 ).
Minimal cardiomyopathy was reported to have increased in a dose-related fashion in male rats but was not considered to have been toxicologically important. In part because this is a common observation in control rats (Greaves et al., 2007b), and, additionally because the incidence was within the historical control range of the laboratory, the severity was not greater than that seen in the control groups, and because these microscopic observations were not associated with any other gross or clinical findings.
Other changes included higher mean thyroid/parathyroid weights with corresponding epithelial hypertrophy and cytoplasmic vacuolation. The histologic changes were mostly judged as minimal. It is plausible that these changes reflected a compensatory response related to the increased metabolic capacity of the liver and more rapid turnover of thyroid hormones (Curran, 1991; Capen, 1997). Lymphoid depletion of the thymus was observed in the high dose females and microscopic findings of cytoplasmic vacuolation of the adrenal cortex were noted in the males and high dose group females. The lymphoid cortical depletion of the thymus and adrenal cortex vacuolation were considered to have been stress responses (Greaves, 2007b), although cytoplasmic vacuolation of the adrenal cortex can also occur spontaneously (Frith et al., 2000) or as the result of pharmacological effects (Greaves, 2007c). The overall no effect level for all systemic effects was 100 mg/kg/day.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

Neurotoxicity

Effect level:

>= 900 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

_Table 2. Results of investigations of hematology and clinical chemistry parameters which were statistically different from control values.

Parameter Measured	Corn Oil Control	100 mg/kg/day	300 mg/kg/day	900 mg/kg/day
Males, data taken at terminal sacrifice
Red Blood Cell Count (10^6/ul)^b	9.22+0.54	9.28+0.28	8.91+0.34	8.78+0.22^c
Hemoglobin (g/dL)^b	15.7+0.72	15.8+0.48	15.2+0.54	14.7+0.44^c
Hematocrit (%)^b	48.1+2.4	48.6+1.6	46.5+1.5	45.0+1.8^d
Platelet (10^3/ul)^b	854+151	885+84	803+144	976+87^d
Leukocytes, absolute (10^3/ul)^b	0.02+0.02	0.03+0.02	0.02+0.02	0.04+0.03^a
RDW (%)^b	11.4+0.4	11.5+0.4	11.6+0.4	12.5+0.6^d
HDW (g/dL)^b	2.58+0.10	2.68+0.12	2.76+0.16^c	2.77+0.27^c
Females, data taken at termination (lactation day 4)
White blood cell count^b	5.15+1.30	6.89+1.58	7.68+2.24^c	7.59+1.85^c
APTT (seconds)^b	16.8+1.9	15.9+2.3	15.8+3.1	13.9+1.4^c
Lymphocytes, absolute (10^3/ul)	3.32+0.61	4.50+1.42	5.11+1.75^c	4.96+1.60^c
Monocytes, absolute (10^3/ul)	0.11+0.10	0.24+0.21	0.21+0.12	0.35+0.23^c

1. Parameters not affected by treatment included:

a.      Males – white blood cell count, mean corpuscular volume (fL), mean corpuscular hemoglobin (pg), mean corpuscular hemoglobin content (g/dL), prothrombin time (sec), APTT (sec), reticulocytes (%), reticulocytes, absolute (10³/ul), MPV (fL), neutrophils (%), lymphocytes (%), monocytes (%), eosinophils (%), basophils (%), leucocytes(%), neutrophils, absolute (10³/ul), lymphocytes, absolute (10³/ul), monocytes, absolute (10³/ul), eosinophils, absolute (10³/ul), basophils, absolute (10³/ul).

b.      Females – red blood cell count (10⁶/ul), Hemoglobin content (g/dL), hematocrit (%), mean corpuscular volume (fL), mean corpuscular hemoglobin (pg), mean corpusc ular hemoglobin content (g/dL), platelet count (10³/ul), prothrombin time (sec), reticulocytes (%), reticulocytes, absolute (10³/ul), ), MPV (fL), neutrophils (%), lymphocytes (%), monocytes (%), eosinophils (%), basophils (%), leucocytes(%), neutrophils, absolute (10³/ul), eosinophils, absolute (10³/ul), basophils, absolute (10³/ul), Leukocytes absolute (10³/ul), RDW (%), HDW (g/dL)

Table3. Statistically significant changes in terminal body weights and organ weights (g). The data are given as mean+SD.

Parameter	Sham Control	Corn Oil Control	100 mg/kg/day	300 mg/kg/day	900 mg/kg/day
Males
Final Body Weight	467+27	454+45	448+45	439+34	412+28
Liver	15.61+1.43	13.46+2.01	13.98+2.04	15.69+1.83*	19.94+2.08^b
Kidney	3.51+0.25	3.21+0.20^a	3.38+0.39	3.53+0.33	3.77+0.46^b
Heart	1.46+0.09	1.46+0.21	1.41+0.14	1.43+0.13	1.32+0.13
Thyroid/parathyroid	0.019+0.002	0.019+0.001	0.020+0.002	0.020+0.002	0.020+0.002
Epididymis (LT)	0.57+0.14	0.60+0.05	0.60+0.04	0.66+0.05^a	0.63+0.06
Epididymis (RT)	0.62+0.04	0.62+0.06	0.61+0.03	0.66+0.04	0.65+0.06
Females
Final body Weight	335+25	313+23	301+30	294+24	289+24
Liver	13.6+2.0	11.7+1.5	12.1+1.1	13.3+1.5	17.9+2.4^b
Kidney	2.39+0.17	2.07+0.18^a	2.11+0.15	2.05+0.25	2.17+0.19
Heart	1.21+0.23	1.10+0.10	1.08+0.10	1.07+0.11	1.01+0.13
Lungs	1.36+0.13	1.40+0.13	1.26+0.12^a	1.20+0.12^b	1.20+0.07^b
Uterus/Vagina	1.07+0.19	1.00+0.14	0.86+0.08^a	0.88+0.11^a	0.85+0.12^a

a = P < 0.05, b = P < 0.01

Table 4. Summary of microscopic findings from rats following repeated treatment with refined naphthenic acids.

Sex	Males				Females
Doses,mg/kg/day	Corn Oil	100	300	900	Corn Oil	100	300	900
N	12	12	12	12	9	12	10	10
Kidney
Hyaline Droplets	0	3	10^b	11^b	0	0	0	0
Minimal	0	3	9^b	9^b	0	0	0	0
Mild	0	0	1	2
Nephropathy	0	0	2	9^b	0	0	0	0
Minimal	0	0	2	5^a	0	0	0	0
Mild	0	0	0	4
Liver
Hypertrophy, hepatocellular, centrilobular	0	0	0	8^b	0	0	0	10^b
Minimal	0	0	0	8^b	0	0	0	10^b
Vacuolation, hepatocellular	2	1	2	0	0	1	0	2
Minimal	1	1	2	0	0	1	0	2
Mild	1	0	0	0	0	0	0	0
Thymus
Depletion, lymphoid, cortex	0	0	0	0	0	1	0	5^a
Minimal	0	0	0	0	0	1	0	4
Mild	0	0	0	0	0	0	0	1
Thyroid
Hypertrophy, epithelial	0	6^a	9^b	11^b	0	3	4	8^b
Minimal	0	6^a	9^b	11^b	0	3	4	6^a
Mild	0	0	0	0	0	0	0	2
Vacuolation, cytoplasmic	0	6^a	9^b	10^b	0	3	4	8^b
Minimal	0	6^a	9^b	10^b	0	3	4	8^b
Adrenal Cortex
Vacuolation, cytoplasmic	0	2	3	2	0	0	0	2
Minimal	0	2	3	2	0	0	0	1
Mild	0	0	0	0	0	0	0	1
Heart
Cardiomyopathy
Minimal	8	5	4	4	3	3	2	4
Mild	4	7	8^a	8^a	0	0	0	0

a = P < 0.05, b = P < 0.01

Conclusions:

The overall no adverse effect level for all systemic effects was 100 mg/kg/day.
The overall no adverse effect level for neurotoxicity was 900 mg/kg/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats was performed by oral gavage with naphtenic acids in corn oil. There were 3 test material treated groups (100, 300 and 900 mg/kg bw) along with a vehicle treated group (corn oil) each in 12 animals/sex/group. Male rats were dosed during premating, mating and afterwards for 28 days in total and females were dosed during premating, mating, gestation and up to day 3 post partum. In this section, only repeated dose toxicity parameters are discussed: (further info on reproductive & developmental parameters is given in Section 7.8.1 and 7.8.2.

At the lowest dose of 100 mg/kg bw, no effects were observed both in male and female rats. At the 300 and 900 mg/kg bw doses, there were no relevant effects on clinical chemistry (only slight incidental findings) and neurobehaviour at all dose levels. Further effects at the medium and high doses are described:

-  Two mortalities and clinical observations were seen in high dose females; clinical observations in some high dose group males.

-   Body weight gain at the high dose was reduced for less than 10% in males and 4% in females versus control groups (not statistically significant). This was associated with reduced food consumption in both groups (statistically significant).

-   Slightly reduced hemoglobin in males (statistically significant), however not in females.

-   Increased liver weight in males and females of medium and high dose groups (only statistically significant at high dose); significant increase in kidney weight in males of medium and high dose group (only statistically significant at high dose).

-   Pale kidneys in the high dose males and reduction in the number of corpora lutea in the high dose females.

-   Hyaline-droplets in high dosed male kidneys (not relevant for humans); liver hepatocellular hypertrophy in high dosed males & females (considered to be rather adaptive than adverse); ad thyroid gland epithelial hypertrophy and cytoplasmatic vacuolation in (mid) and high dosed males & females (considered to be compensatory related to the increased metabolic capacity of the liver and more rapid turnover of thyroid hormones).

In conclusion, NOAEL for systemic toxicity was 100 mg/kg bw, whereas NOAEL for neurotoxicity was 900 mg/kg bw, both in male and female rats.

Data source

Reference

Reference Type:

other: robust study summary

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Naphthenic acids

EC Number:

215-662-8

EC Name:

Naphthenic acids

Cas Number:

1338-24-5

Molecular formula:

For the acidic (naphthenic) fraction: CnH2n+zO2, where n = carbon number and z = homologous group series number: z = 0 when no ring structures are present, z = -2 when 1 ring is present, z = -4 when 2 rings are present etc.

IUPAC Name:

3-(3-ethylcyclopentyl)propanoic acid

Test material form:

other: liquid

Details on test material:

The test sample used in the current program was a blend of naphthenic acids from three sources. The samples were dried under a stream of nitrogen and then re-dissolved in 0.5 mL dichloromethane. The samples were analyzed by GC-MS (Young et al., 2008) and the total ion current mass spectra were collected and tablulated (Holowenko et al., 2002).

Based on these data it was determined that there were no significant differences among these samples (Fedorak, 2009). The data indicated that the test material contained constituents with carbon numbers predominantly in the range of C6-C16 (corresponding to a molecular weight range of approximately 116-250) and with a ring distribution of approximately 0 rings (24%), 1 ring (39%), 2 rings (31%), 3 rings (5%) and 4 rings (1%).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The naphthenic acids were suspended in corn oil to the appropriate concentrations and administered in 10 ml/kg doses.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days. All females confirmed to have mated were placed in plastic maternity cages once mating was confirmed.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Females for which copulation was not detected were placed in maternity cages at the end of the 1
- Any other deviations from standard protocol:Length of gestation was calculated as the time from confirmation of mating to the onset of delivery.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Dosing of males was initiated 14 days prior to pairing and throughout a 14 day mating period for a total of 28 - 29 doses. Dosing of females was also initiated 14 days prior to pairing and continued throughout the mating and gestational periods until study termination on post-natal day 3. The total number of doses ranged from 39 - 53 depending on the time at which mating occurred.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- All rats were examined twice daily for mortality and general health.
- All animals were examined approximately 1 hour after each treatment, and all unusual observations were recorded.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Detailed physical examinations of all animals were conducted weekly (See Section 7.5.1)

BODY WEIGHT: Yes
- Time schedule for examinations: females: recorded once week prior to test substance administration, on the first day of dose administration and weekly until evidence of copulation was obtained. From that point body weights of female rats were recorded on gestation days (GD) 0, 4, 7, 11, 14, 17, and 20 and on lactation days (LD) 0, 1 and 4 (termination). For females for which there was no evidence of copulation, body weights were recorded weekly until termination.

FOOD CONSUMPTION:
- Food consumption by adult animals was also recorded on the same schedule as the body weights.

WATER CONSUMPTION: No

OTHER:
- Parental mating, fertility, conception and copulation indices , gestation length, numbers of former implantation sites, absolute and relative organ weights, and pre-coital intervals.
- Toxicological parameters: See Section 7.5.1.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
On the day of parturition, all pups were examined for viability, for the presence of gross malformations and to assess gender. The numbers of live and stillborn pups were recorded.
All offspring were uniquely identified and examined daily for signs of mortality and ill health. All offspring were individually weighed on PND 1 and 4. Gender was assessed on PND 0 and 4. At scheduled termination, PND 4, all surviving offspring were euthanized and discarded without further examination.

GROSS EXAMINATION OF DEAD PUPS: no data

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: 14 days after mating
- Maternal animals: Females for which there was no evidence of mating were sacrificed on post-cohabitation day 25, those that showed evidence of mating but failed to deliver were euthanized on post-mating day 25, and all others were euthanized on post-natal day 4.

GROSS NECROPSY: YES
- At termination rats were euthanized by carbon dioxide inhalation.
- Necropsies were conducted on all animals sacrificed in extremis or at study termination.

HISTOPATHOLOGY / ORGAN WEIGHTS
- An examination of target organs including male and female reproductive organs was also carried out as part of this test (See also Section 7.5.1).
- Organs examined included: ovaries with oviduct, uterus with cervix and vagina, testes with epididymides, prostate and seminal vesicles.
- The ovaries, testes and uteri were weighed and all were examined histologically

Postmortem examinations (offspring):

All offspring were individually weighed on PND 1 and 4.
Gender was assessed on PND 0 and 4. At scheduled termination, PND 4, all surviving offspring were euthanized and discarded without further examination.

Statistics:

Parental mating, fertility, conception and copulation indices were analysed using the Chi-square test with Yates’ correction (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites, numbers of corpora lutea, number of pups born, live litter size on PND 0, unaccounted for sites, absolute and relative organ weights, and pre-coital intervals were evaluated by one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences between the vehicle control and test substance-treated groups. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group.

Reproductive indices:

See Tables below and in Section 7.8.2.
Mating, fertility, pregnancy and gestation indices were not provided as such, however No. of females mated, pregnant and with litters were given.
Pre-implantation loss not provided, but No. of corpora lutea and No. of implantation sites given.
Implanation index not provided, but No. of implanatation given.
Post-implantation indexes given.

Offspring viability indices:

Viability index not provided, but No. born and alive on day 4 given.
Sex ratio given (See Section 7.8.2)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

The absolute epididymal weights were increased in the 900 mg/kg/day group but were not significantly different when expressed on a per body weight basis. The uterine weights were also significantly elevated but this was considered to have been a consequence of the fact that the females were all in lactational anaestrous. The uterine weights were within the historical range of the laboratory and were not considered to have been toxicologically important. There were no weight differences in any of the other organs and no pathological changes in any of the reproductive organs at the highest dose tested (900 mg/kg/day).

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: mating index

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: reproductive organ effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Decrease No. born pups at 900 mg/kg bw - See Section 7.8.2

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decrease at 900 mg/kg bw - See Section 7.8.2

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 900 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Summary of reproductive parameters assessed in the repeated dose/reproductive toxicity study of refined naphthenic acids.

Dose (mg/kg/day)	Corn Oil Control	100 mg/kg/day	300 mg/kg/day	900 mg/kg/day
Number of females paired	12	12	12	12
Number of female mated	12	12	10	11
Number of females pregnant^a	9	12	10	11
Number of females with litters	9	12	10	11
Pre-coital interval (days)^b	1.4+0.7	2.3+1.1	4.2+3.3^c	3.8+3.5
Gestation length (days)	21.4+0.6	21.9+0.3	22.0+0.5	22.1+0.5
Corpora lutea	15.6+2.3	14.0+1.4	15.1+3.0	13.8+2.1
Implantation sites	15.0+2.4	13.6+1.1	13.0+1.2	12.2+3.7
Number born	14.1+1.9	12.9+1.1	12.0+1.6	10.8+3.8^c
Post-Implantation loss (%)^d	6.0	5.1	7.7	11.5

a. Pregnant = uterine implantation sites.

b. Data summarized as mean+standard deviation.

c. p < 0.05

d. Post-implantatoin loss = (No. of implantations - No. of life fetuses)/No. of implanatations (%)

Applicant's summary and conclusion

Conclusions:

No reproductive effects were identified. The NOAEL for mating and reproductive effects is 900 mg/kg/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats was performed by oral gavage with naphthenic acids in corn oil. There were 3 test material treated groups (100, 300 and 900 mg/kg bw) along with a vehicle treated group (corn oil) each in 12 animals/sex/group. Male rats were dosed during premating, mating and afterwards for 28 days in total and females were dosed during premating, mating, gestation and up to day 3 post partum. In this section, only reproductive toxicity parameters are discussed: (further info on repeated & developmental parameters is given in Section 7.5.1 and 7.8.2. Target organ findings to P0 group were identified at the dose of 900 mg/kg bw, whereas 100 mg/kg bw was considered as the NOAEL for systemic toxicity.

No reproductive effects were identified up to 900 mg/kg bw. There were no weight differences in any of the other organs nor any pathological changes in the reproductive organs up to the highest dose tested (900 mg/kg/day). The NOAEL for mating and reproductive organ effects was 900 mg/kg/day.