Naphthenic acids, cobalt salts

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-04-27 to 2008-11-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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)

Version / remarks:

1996-03-22

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Neodecanoic acid, cobalt salt

EC Number:

248-373-0

EC Name:

Neodecanoic acid, cobalt salt

Cas Number:

27253-31-2

Molecular formula:

Molecular and structural formula as well as molecular weight are not applicable to a UVCB substance.

IUPAC Name:

Neodecanoic acid, cobalt salt

Test material form:

other: paste

Details on test material:

- Name of test material (as cited in study report): Neodecanoic Acid, Cobalt Salt
- State of aggregation: blue/purple thick liquid, maybe semi-solid

Test animals

Species:

rat

Strain:

other: Crl: CD(SD)

Details on species / strain selection:

The rat was selected for this study because it is a preferred species for toxicity testing as recommended by test guidelines. The Crl:CD(SD) strain was chosen because extensive background information is available from the literature, the supplier, and previous studies conducted by the laboratory. This strain is also considered suitable relative to hardiness and incidence of spontaneous disease.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Females nulliparous: yes
- Age at the start of treatment: males: approximately 65 days; females: approximately 71 days
- Weight at the start of treatment: males: 301.9 - 362.0 g; females: 204.4 - 260.5 g
- Housing:
males (pretest, premating period and postmating): housed individually in stainless, wire-mesh cages suspended above cageboards.
males (cohabitation period): housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboards.
females (cohabitation period): housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboards.
females (without evidence of copulation, postmating): housed individually in polycarbonate pans.
females (evidence of copulation; Days 0 - 19 of gestation): housed individually in stainless steel, wire-mesh cages suspended above cageboards.
females (evidence of copulation; Day 20 of gestation - Day 4 of lactation): housed individually in polycarbonate pans with bedding.
females (lactation period): housed with their litters in polycarbonate pans with bedding.
- Diet (ad libitum, except when fasted): pelleted PMI® Nutrition International, Certified Rodent LabDiet® 5002
- Water (ad libitum): tap water
- Quarantine period: approximately 7 days

DETAILS OF FOOD AND WATER QUALITY:
- water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants.
- certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 ºC (target with an acceptable tolerance range of 18-26 ºC)
- Relative humidity: 30 %–70 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Mazola®

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- formulations of the test substance in the vehicle were prepared daily and stored at room temperature until used.
- volume of test substance or vehicle given to each rat was based on the most recently recorded body weight.
- dosing volume: 5 mL/kg

VEHICLE
- corn oil was not expected to contain any contaminants that would have interfered with the conduct of the study.
- vehicle was assumed to be stable under the conditions of the study and was stored at room temperature.

Details on mating procedure:

- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: until evidence of copulation was observed or until a period of 2 weeks had elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): On the day copulation was confirmed, the female was transferred back to individual cage housing.
Days 0 - 19 of gestation: stainless steel, wire-mesh cages suspended above cageboards
Day 20 of gestation - Day 4 of lactation: polycarbonate pans with bedding

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation were collected once near the beginning of the study from all the formulation concentrations, and again near the end of the study. One sample of the vehicle and 2 samples from each formulation concentration were collected. Samples from dosing formulations were submitted for concentration verification analysis. Concentrations of the cobalt neodecanoate in separate dosing formulation samples were measured by inductively coupled plasma (ICO) spectroscopy.

Results:
The recovery values obtained for these samples were 98 - 104 %
The data for samples collected indicate that the test substance was at the targeted concentrations in the samples (± 20 % of nominal) up to eight days after formulation. Cobalt neodecanoate was not detected in the blank.

Duration of treatment / exposure:

Males: 47 and 48 days
Non-pregnant females/pregnant females: 41-48 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12 males / 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a two-week range finding study (DuPont Haskell (2006a))*, male and female rats were dosed with 30, 300, or 600 mg/kg/day of the test substance (dose volume: 10 mL/kg). The 300 and 600 mg/kg/day groups were terminated due to mortality, clinical signs of toxicity, and body weight loss. At 30 mg/kg/day, mean final body weight and overall body weight gain were reduced in males (20 % and 70 % lower than the control group, respectively) and females (3% and 31% lower than the control group, respectively). Clinical signs of toxicity were observed in males only at 30 mg/kg/day.
Additional male rats were added to the study design and were dosed with 5 or 15 mg/kg/day of the test substance (dose volume: 5 mL/kg). There were no clinical signs of toxicity at any dose level tested. Final body weights were 4 % lower than controls at 15 mg/kg/day. Overall mean body weight gain was 22 % lower than controls at 15 mg/kg/day. There were no reductions in final body weight or overall body weight gains at 5 mg/kg/day.
In a study with a similar cobalt containing compound (DuPont Haskell (2006b))*, decreasing the dose volume from 10 to 5 mL/kg resulted in significantly reduced toxicity at 30 mg/kg/day. Based on these results, the concentrations selected for the current study were 0, 5, 15, and 45 mg/kg/day.

*References:
- DuPont Haskell (2006a). Repeated Dose Oral Toxicity: 2-Week Gavage Study in Rats. Unpublished Report. DuPont Haskell HL- 27601
- DuPont Haskell (2006b). Repeated Dose Oral Toxicity: 2-Week Gavage Study in Rats. Unpublished Report. DuPont Haskell HL- 27601

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
1) daily animal health observations: at least once daily during the quarantine/pretest period and once daily during the dosing period
2) careful clinical observations: within 3 hours post-dosing; on 2 separate occasions, observations were performed approximately 4 to 5 hours after dosing.
- Cage side observations checked:
1) daily animal health observations: mortality/moribund and abnormal behavior and/or appearance
2) careful clinical observations: acute/systemic toxicity
Starting on day 20 of gestation for mated females or 7 days after the end of the cohabitation period for females without evidence of copulation, female rats were observed at least twice daily for signs of delivery and offspring.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before first dosing (baseline), and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
1) females
- premating period: once a week
- gestation period: days 0, 7, 14, and 21 of gestation.
- lactation period: days 0 and 4 of lactation
- females without evidence of copulation as well as those that copulated and did not deliver a litter: weekly schedule until sacrifice

2) males:
weekly schedule until sacrifice

All rats that survived to the scheduled terminal sacrifice were weighed prior to sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Each feeder was weighed at the beginning and end of the weekly food consumption interval and the final weight of the feeder and the amount of spillage from the feeder during the interval were subtracted from the initial feeder weight. From the food consumption and body weight data, the mean daily food efficiency was calculated.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
Premating and cohabitation periods: individual food consumption was determined weekly throughout the period, ending on test day 14. Food consumption was not measured during cohabitation for males and females or after cohabitation for males or females without evidence of copulation.
Gestation and lactation periods: individual food consumption of presumed pregnant P1 females was determined on gestation days 0, 7, 14, and 21, and for lactating females on lactation days 0 and 4. Food consumption was not measured for females that did not deliver a litter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: Yes
- mean daily food efficiency (g weight gain/g food consumed)

WATER CONSUMPTION AND COMPOUND INTAKE: No

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Parameters examined in male parental generations:
- organ weights: testis, epididymis, prostate, seminal vesicles (with coagulating glands and fluids)

Litter observations:

The day when delivery was complete was designated day 0 postpartum.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Day 0 postpartum:
- abnormality, abnormal behaviour, appearance, and mortality
- number and sex of live and dead pups of each litter
- body weight of live pups of each litter
Day 4 postpartum:
- abnormality, abnormal behaviour, appearance, and mortality
- number and sex of live pups of each litter
- body weight and gross external examination of live pups of each litter

GROSS EXAMINATION OF DEAD PUPS:
A gross postmortem examination was conducted for pups that were moribund, found dead, or sacrificed because of death of the dam during lactation.

Postmortem examinations (parental animals):

SACRIFICE
Rats that survived to the scheduled sacrifice were euthanized on days 48–49 (males) or days 42–49 (females). All animals were dosed until the day prior to sacrifice. Rats with poor health were submitted for euthanasia prior to the scheduled sacrifice.

GROSS NECROPSY
Gross examinations were performed on all adult rats. The presence and number of uterine implantation sites and ovarian corpora lutea were evaluated for all cohabited females.
Following tissues were collected from all adults:
liver, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, urinary bladder, lungs, trachea, heart, bone marrow (collected with sternum), thymus, spleen, mandibular lymph node, mesenteric lymph node, Peyer’s patches (collected from sections of the intestine), thyroid gland, adrenal glands, brain (3 sections, including cerebrum, cerebellum, midbrain and medulla/pons), spinal cord (3 sections, including cervical, mid-thoracic, and lumbar spinal cord), sciatic nerve, sternum, testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviducts, uterus, cervix, vagina, and gross observations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were weighed wet from all adult rats that survived to the scheduled sacrifice:
liver, kidneys, heart, thymus, spleen, adrenal glands, brain, testes, epididymides, prostate, seminal vesicles (with coagulating glands and fluids), ovaries (with oviducts), and uterus (with cervix). Paired organs were weighed together. Organ weight ratios (% final body weight, % brain weight) and group mean values were calculated.

All tissues were fixed, processed, sectioned, stained, and examined microscopically.
Processing and microscopic evaluation of tissues were as follows:
- 45 mg/kg/day dose group females: only one female rat in the 45 mg/kg/day group survived to the scheduled sacrifice. Tissues from animals in this group were not evaluated microscopically.
- scheduled sacrifice: all tissues were evaluated microscopically from up to 5 rats/sex that were sacrificed by design in the control and 45 mg/kg/day dose group (males only) and 15 mg/kg/day dose group (females only).

- Decedents: all protocol tissues were evaluated microscopically from rats that were submitted for an unscheduled sacrifice due to deteriorating health and for rats that were found dead.

- Reproductive failures: all reproductive tissues were evaluated microscopically from the male and female mated pairs that failed to produce a litter.

- Target organs: available target organs were evaluated microscopically from all adult rats in all groups (males: spleen and bone marrow; females: intestinal tract (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum), spleen, thymus, adrenal glands, and kidneys).

Postmortem examinations (offspring):

SACRIFICE/GROSS NECROPSY
A gross postmortem examination was conducted for pups that were moribund, found dead, or sacrificed because of death of the dam during lactation.

HISTOPATHOLOGY / ORGAN WEIGTHS
Organs were not weighed and no microscopic examinations were conducted on any F1 pups.

Statistics:

The following statistical tests were used
- Levene’s test for homogeneity
- Shapiro-Wilk test for normality
- One-way analysis of variance
- Dunnett’s test
- Kruskal-Wallis test
- Dunn's test
- Sequential application of Cochran-Armitage test for trend
- Analysis of covariance
- Dunnett-Hsu- Non-parametric analysis of covariance
- Repeated measures analysis of variance followed by Linear contrasts
- Sequential application of the Jonckheere-Terpstra trend test
- Fisher’s Exact test with a Bonferroni correction
Male and female data were evaluated separately. For litter parameters, the proportion of affected pups per litter or the litter mean were used as the experimental unit for statistical evaluation (Haseman and Hogan, 1975).
* The level of significance selected was p < 0.05.

*Reference:
- Haseman, J.K. and Hogan, M.D. (1975). Selection of the experimental unit in teratology studies. Teratology, 12, 165-171.

Reproductive indices:

Mating index (%) = (number mated*/number paired) x 100
Fertility index (%) = (number pregnant**/number mated*) x 100
Post-implantation Loss (%)*** = ((number of implantation sites - number of pups born)/number of implantation sites) x 100

*evidence of copulation = intravaginal copulatory plug, sperm in vaginal lavage, uterine implantation sites, or delivery of a litter.
** including those found dead pregnant during gestation.
*** determined for each litter.

Offspring viability indices:

Live born index (%)*= (number of pups born alive / number of pups born) x 100
Viability index (%)** = (number of pups alive PND 4 preculling / number of pups born alive) x 100

* determined for each litter.
** determined for each litter.; excluding litters sacrificed due to death of dam during lactation

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males:
- 15 and 45 mg/kg/day: test substance-related clinical signs of toxicity occurred which included wet fur in the perioral/chin region following the daily dosage administration (not considered to be adverse).
Females:
Gestation:
- 15 and 45 mg/kg/day: test-substance-related clinical signs of toxicity were observed. Females had increased incidences of labored breathing, dehydration, diarrhea, dystocia, and hunched over posture during the daily post-dosing observations.
Lactation:
- 5, 15, and 45 mg/kg/day:
test-substance-related clinical signs of toxicity were observed, as follows:
- 45 mg/kg/day group: lethargy, stained fur/skin (yellow, red, and/or brown), ataxia, and slow breathing rate, during the daily post-dosing observations or weekly detailed clinical observations.
- 15 mg/kg/day group: yellow stained fur/skin.
- 5 mg/kg/day group: lethargy and yellow stained fur/skin.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Females:
Gestation:
- 15 and 45 mg/kg/day: test substance-related mortality occurred. Seven rats in the 45 mg/kg/day group were found dead during gestation days 21-22, and 2 were sacrificed in extremis on gestation day 22. Two rats in the 15 mg/kg/day group were found dead during gestation days 22-23, and 3 were sacrificed in extremis on gestation days 22-23.
Lactation:
- 5 or 45 mg/kg/day: test substance-related mortality occurred. One female in the 5 mg/kg/day group was found dead on lactation day 1. One female in the 45 mg/kg/day group was found dead on lactation day 1, and one was sacrificed in extremis on lactation day 1. Number of dams surviving to lactation day 4 was 11, 11, 6, and 1 for the 0, 5, 15, and 45 mg/kg/day groups, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 5, 15, or 45 mg/kg/day: adverse, test substance-related, statistically significant decreases in body weight and/or weight gain occurred.
- 45 mg/kg/day: body weight was significantly decreased during test days 27, 34, 41, and 48, and weight gain was decreased over the intervals of days 0-7, 13-20, 20-27, 27-34, 34-41, 41-48, 0-13, 13-48, and 0-48. By test day 48, males had 18% lower body weight, and weight gain over days 0-48 was 50% lower than the control value.
- 15 mg/kg/day: body weight was slightly lower (8%) on test day 48, and weight gain was significantly lower over the interval of test days 34-41. Body weight gain values over the intervals of test days 13-48, and 0-48 were significantly lower (37% and 25%, respectively) compared to the control value.
- 5 mg/kg/day: body weight gain was significantly lower for the interval of test days 13-48 (18%) compared to the control value; weight gain for the interval of test days 0-48 was 12% lower than the control value (not statistically significant).

2) Females:
Gestation:
- 15 and 45 mg/kg/day: adverse, test substance-related, statistically significant decreases in body weight and weight gain occurred.
- 45 mg/kg/day: on gestation day 21, body weight was 21% lower and weight gain values for gestation days 14-21 and 0-21 were 68% and 49% lower than the control values, respectively.
- 15 mg/kg/day: body weight was 12% lower on gestation day 21, and weight gain values for gestation days 14-21 and 0-21 were 46% and 29% lower than the control values, respectively.
Lactation:
- 45 mg/kg/day: adverse, test substance-related decreased body weight was observed on lactation day 0; weight gain during lactation days 0-4 in the one surviving dam was similar to control.

Please also refer for results to the field "Attached background material" below.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 45 mg/kg/day: test substance-related, statistically significant decreases in food consumption occurred. Food consumption was significantly decreased during test days 0-7.

2) Females:
Gestation:
- 15 and 45 mg/kg/day: adverse, test substance-related, statistically significant decreases in food consumption and food efficiency occurred.
- 45 mg/kg/day: food consumption values were significantly lower on gestation days 7-14, 14-21, and 0-21, and were 20% lower than the control value for gestation days 0-21.
- 15 mg/kg/day: food consumption and/or food efficiency was significantly lower for gestation days 14-21 and gestation days 0-21, and was 21% and 10% lower than the control values for gestation days 14-21 and 0-21, respectively.
Lactation:
- 15 and 45 mg/kg/day: adverse, test substance-related, decreases in food consumption occurred. Food consumption was 21% and 35% lower than the control value during lactation days 0-4 in 15 and 45 mg/kg/day females, respectively.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 45 mg/kg/day: test substance-related, statistically significant decreases in food efficiency occurred. Food efficiency was significantly decreased during test days 0-7, and food efficiency was decreased during test days 0-13. Since the food efficiency value over test days 0-13 was only 7% lower than the control value, this minimal difference was not considered to be adverse.

2) Females:
Gestation:
- 15 and 45 mg/kg/day: adverse, test substance-related, statistically significant decreases in food efficiency occurred.
- 45 mg/kg/day: food efficiency values were significantly lower for gestation days 7-14, 14-21, and 0-21, and were 38% lower than the control value for the gestation days 0-21 period.
- 15 mg/kg/day: food consumption and/or food efficiency was significantly lower for gestation days 14-21 and gestation days 0-21, and was 21% and 10% lower than the control values for gestation days 14-21 and 0-21, respectively.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Males:
- 45 mg/kg/day: adverse, test substance-related, statistically significant decreases in mean duration of movement and number of movements occurred. Total duration of movement was 22% lower compared to the control value, and mean number of movements was 12% lower compared to the control value. Mean duration of movement and number of movements were also significantly lower during the sixth 10-minute interval. In the absence of other neurobehavioral changes, the decreased motor activity was considered to be secondary to systemic toxicity (gastroenteropathy, dehydration, reduced body weight and body weight gain, reduced food consumption).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 45 mg/kg/day: test substance-related microscopic findings were limited to increased erythropoiesis in the spleen and bone marrow. Minimally increased extramedullary hematopoiesis of the spleen was present in 4/12 rats and minimal erythroid hyperplasia of the bone marrow was present in 9/12 rats. Neither findings were present in the remaining treated groups or in the control groups.
- increased erythropoiesis in bone marrow and spleen was correlative to the slight increase in red cell mass parameters, was not associated with evidence of toxicity in these organs, and thus was not considered adverse.

2) Females (tissues from females in the 45 mg/kg/day group were not evaluated microscopically):
- 5 and 15 mg/kg/day: test substance-related microscopic findings were present, as summarized below:
Gastrointestinal tract:
- several degenerative and regenerative changes, as well as mucosal atrophy, were present in the gastrointestinal tract of 8/12 rats in the 15 mg/kg/day, and in one dead rat in the 5 mg/kg/day group. These gastrointestinal changes were interpreted to be the cause of death in all female decedents.
- 3/7 rats in the 15 mg/kg/day group that survived to the terminal sacrifice had test substance-related microscopic changes in the gastrointestinal tract, which were generally minimal to mild and limited to only one section of the gastrointestinal tract.
- changes observed in the intestines included the following diagnoses: degeneration/necrosis of mucosal epithelium (villous and crypt epithelium); atrophy of villi and crypts; regeneration of mucosal epithelium, and mucosal inflammation. Lesions were observed in all segments of the small intestine and in the cecum and colon. Lesions were graded as minimal to severe and were most severe in the jejunum, ileum, and cecum.

- further changes:
15 mg/kg/day: low incidences of erosion or ulceration of the gastric mucosa were present. Ulcer/erosion was present in the glandular mucosa of 3/12 rats and in the nonglandular mucosa of 2/12 rats. Goblet cell depletion of the rectum was present in 4/12 rats. 5 mg/kg/day: goblet cell depletion of the rectum was present in one rat (the decedent).

Lymphoid organs:
- 15 mg/kg/day: test substance-related necrosis of lymphocytes and/or atrophy of lymphoid regions were present in the thymus and/or spleen in 9/12 rats.
- 5 mg/kg/day: lymphoid atrophy was present in 4/12 rats. Lymphoid atrophy was limited to the thymus, except for one animal which also had splenic involvement.
- lymphoid changes were graded as minimal to severe and were generally most consistently present and most severe in the thymus.

Adrenal glands:
- 15 mg/kg/day: minimally increased microvesiculation of adrenal cortical cells was present in 5/12 rats. The change was diffuse within the cortex and was characterized by multiple, small, clear vacuoles within the cytoplasm of adrenal cortical cells. These changes in the current study were considered to be test substance related, but they were not considered adverse, as they were not associated with morphologic evidence of cytotoxicity to affected cells.

Kidneys:
- 15 mg/kg/day: minimal vacuolation of renal tubular epithelium was present in renal cortical tubules of 5/12 rats. Tubular vacuolation was characterized by small, discrete, clear granules in the basilar cytoplasm of renal cortical epithelial cells. This change was not associated with other microscopic findings or with clinical pathology changes indicative of renal injury and vacuolation of renal tubules was not considered to be adverse.
- 5 mg/kg/day: minimal vacuolation of renal tubular epithelium was present in renal cortical tubules of the one decedent. Tubular vacuolation was characterized by small, discrete, clear granules in the basilar cytoplasm of renal cortical epithelial cells. This change was not associated with other microscopic findings or with clinical pathology changes indicative of renal injury and vacuolation of renal tubules was not considered to be adverse.

- all other microscopic findings in this study were consistent with normal background lesions in rats of this age and strain.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
1) Males:
- no unscheduled deaths in any male group.
- the remainder of the clinical observations recorded besides the ones stated above was common for this age and strain of rat, and was not considered to be test substance related.

2) Females:
Premating:
- test substance-related mortality or clinical observations did not occur

BODY WEIGHT AND WEIGHT GAIN
2) Females:
Premating:
- 45 mg/kg/day: body weight gain was slightly decreased (31% lower compared to the control value, not statistically significant) over the interval of test days 0-13.
- 5 or 15 mg/kg/day: no test substance-related or statistically significant differences.
Gestation:
- 5 mg/kg/day: no test substance related effects or statistically significant differences on body weight parameters.
Lactation:
- 5 or 15 mg/kg/day: no effects on weight or weight gain during lactation days 0-4.

Please also refer for results to the field "Attached background material" below.

FOOD CONSUMPTION AND COMPOUND INTAKE/FOOD EFFICIENCY
1) Males:
- 5 and 15 mg/kg/day: no test substance-related or statistically significant effects on food consumption or food efficiency

2) Females
Premating:
- no test substance-related or statistically significant effects on food consumption parameters at any level tested.
Gestation:
- 5 mg/kg/day: no test substance-related or statistically significant effects on food consumption or food efficiency.
Lactation:
- 5 mg/kg/day: no test substance-related or statistically significant effects on food consumption or food efficiency.

HAEMATOLOGY
- no adverse changes in mean hematology parameters in male or female rats at test day 14.
The following statistically significant changes were considered non-adverse or not treatment related:
- red blood cell, hemoglobin, hematocrit were minimally increased in male and female rats dosed with 45 mg/kg/day (variable statistical significance; <108% of control). Associated with these minimal increases were minimal but statistically significant increases in absolute reticulocyte counts (180% and 172% of control, male and female) and a minimal increase in red cell distribution width (116% of control, males only). These hematology changes are likely treatment related, but due to the minimal nature of these changes (increases in red cell mass parameters were less than 10% above the control group means), they were not considered to be adverse.
- platelet count was minimally increased in male rats dosed with 15 or 45 mg/kg/day (124% and 135% of control, respectively) (uncertain relationship to treatment; no toxicological significance).
- absolute eosinophil count was minimally decreased in male rats dosed with 5 mg/kg/day (54% of control; not dose related)
- prothrombin time was minimally prolonged in male rats dosed with 45 mg/kg/day (106% of control; no clinical significance).

CLINICAL CHEMISTRY
- no adverse changes in mean clinical chemistry parameters in male or female rats at test day 14.
The following statistically significant changes were considered non-adverse:
- cholesterol was mildly decreased in male rats dosed with 15 or 45 mg/kg/day (variable significance, 70% and 57% of control). Since minimal decreases in cholesterol are not known to be associated with adverse effects in male rats, these changes in cholesterol at 15 and 45 mg/kg/day were not considered to be adverse.
- inorganic phosphorus was minimally increased in male rats dosed with 45 mg/kg/day (110% of control). Values in individual animals were similar to controls, no changes in other parameters that are often associated with increases in inorganic phosphorus, and no treatment-related changes in inorganic phosphorus were present in females. Therefore, the minimal increase in inorganic phosphorus was not considered to be treatment related.
- chloride was minimally increased in male and female rats dosed with 15 or 45 mg/kg/day (103-105% of control). Mean chloride concentrations were similar across all groups, reflecting the lack of relevant change across the range of concentrations of the test substance. The changes were considered to be non-adverse based on their minimal nature.

The following statistically significant changes in group mean clinical chemistry parameters at test day 14 were considered to be unrelated to treatment (and non-adverse) because the change did not occur in a dose-related pattern:
Females:
- alkaline phosphatase was minimally increased and calcium was minimally decreased in rats dosed with 5 mg/kg/day (148% and 95% of control, respectively).
- creatinine was minimally increased in rats dosed with 5 or 15 mg/kg/day (111% and 117% of control, respectively).

NEUROBEHAVIOUR
1) Abbreviated functional observational battery
- 0, 5, 15, 45 mg/kg/day: no test substance-related effects or statistically significant differences on forelimb and hindlimb grip strength in either males or females. No statistically significant effects for any behavioral parameter evaluated in males or females.
- dark colored urine was observed in 3 males administered 45 mg/kg/day and one female in the 15 mg/kg/day group (no adverse effects associated with this observation).

2) Motor activity
Females:
- no test substance-related or statistically significant differences in duration of movement or number of movements at any dosage of the test substance.

ORGAN WEIGHTS
1) Males:
Liver:
- weight parameters (absolute weight, and weight relative to both body and brain weight) had statistically significant decreases relative to controls in all treated groups.
- final body weights taken at terminal sacrifice were also decreased in these groups (statistically significant in the 45 mg/kg/day group).
- since liver weight decreases in association with decreases in body weight, the decreases in liver weight parameters in treated male groups were due, at least in part, to the decreased body weight in these groups.
- conclusion: decreases in liver weights relative to body weight were less severe (compared to control) than were changes in absolute liver weight and liver weight relative to brain weight. Furthermore, liver weight changes were not associated with correlative, treatment-related changes in liver-related clinical pathology parameters or with microscopic changes in the liver. These liver weight changes were not considered to be adverse findings.

2) Females:
- organ weights of the 45 mg/kg/day group were not included in the evaluation, since organ weight data were available for only one female. The organ weights of te early decedents in that group were not collected.
- no other primary test substance-related organ weight changes in male or female rats at any of the dose levels were evaluated.
- several statistically significant organ weight changes in male rats in the 45 mg/kg/day group were considered secondary to mean final body weight change (taken at necropsy) in this group (decreased to 82% of control). No treatment-related microscopic findings were present in affected organs. - for organs whose weight tends to decrease with decreases in body weight (heart, seminal vesicles), statistically significant changes were characterized by decreased absolute weight and organ weight relative to brain weight.
- for organs whose weight is less affected by decreases in body weight (spleen, testes, brain, kidney) statistically significant weight changes were limited to increases in organ weight relative to body weight.
- a statistically significant decrease in brain weight relative to body weight in the 15 mg/kg/day male group was considered to be due to decreased final body weight in this group (body weight decrease not statistically significant).
- a statistically significant increase in heart weight relative to brain weight in the 15 mg/kg/day male group was not considered test substance related as it did not occur in a dose-related manner.
- a statistically significant increase in liver weight relative to body weight in the 15 mg/kg/day female group was not associated with changes in other liver weight parameters or with test substance-related clinical pathology or microscopic changes in the liver. Therefore, this change was not considered to be test substance related.

GROSS PATHOLOGY
1) Males:
- no test substance-related gross observations.

2) Females:
- 45 mg/kg/day: one female had evidence of dystocia, which may have been contributory to its early death.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- no test substance-related effects on mating, fertility, pre-coital interval, or gestation length, or post-implantation loss at any dosage.
- 45 mg/kg/day: number of implantation sites and number of corpora lutea were slightly lower (not statistically significant).
- 5, 15, and 45 mg/kg/day: mortality observed in dams that occurred within the last few days of gestation (gestation days 21-22) and around the time of parturition (lactation days 0-1) is test substance related and appears to be related to that specific phase during pregnancy (mechanism involved could not be discerned)
- precoital interval was statistically higher in 5 mg/kg/day females compared to the control value, but not for the 15 and 45 mg/kg/day groups (no dose response).
- 0 and 15 mg/kg/day: two adult pairs (one pair each in the groups) failed to produce litters.
These incidences of reproduction failures were not dose related and thus was not considered to be test substance related. There were no gross or microscopic pathology findings to explain the reproductive failures.

Effect levels (P0)

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- 45 mg/kg/day: adverse, test substance-related effects on the number of offspring born, born alive, and/or surviving to lactation day 4 occurred. Only 3 litters were born, and the mean number of offspring per litter was 39% lower than the mean number of offspring per litter for the controls. Only one litter survived to postnatal day 4. These effects resulted in significantly lower number of offspring born alive, lower number of offspring alive on postnatal day 4, and lower live born index
- 5 mg/kg/day: 16 offspring from 2 litters were found dead during postnatal day 0-4 (test substance related and adverse effect, given the effects on maternal and offspring survival observed at dosages of 15 and 45 mg/kg/day)
- 45 mg/kg/day: 11 offspring from 2 litters were found dead during postnatal day 0-4
- 0 mg/kg/day: 4 offspring from 2 litters were found dead during postnatal day 0 - 4.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 5, 15, and 45 mg/kg/day: adverse, test substance-related, statistically significant effects on offspring body weight were observed in the 5, 15, and 45 mg/kg/day groups.
- 45 mg/kg/day: of the surviving offspring, mean weights were 5% lower than the control means on postnatal day 0.
- 15 mg/kg/day: offspring body weight was significantly lower than the control value on postnatal day 0 and postnatal day 4 (15% and 13%, respectively).
- 5 mg/kg/day: offspring body weight was also 7% lower (statistically significant) than the control group on postnatal day 4.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Most pups submitted for necropsy were from females that were found dead or were sacrificed prior to delivery of their litters. Thus, incidences of lesions commonly seen in term foetus that die or are not delivered were increased in the treated groups. These included unexpanded lungs and absence of a milk spot in the stomach. There were no other test substance-related gross observations in pups.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

CLINICAL SIGNS (OFFSPRING)
- no test substance-related clinical observations in the surviving offspring at any dose level tested.

OTHER FINDINGS (OFFSPRING)
- Sex ratio: ratio was statistically higher in the 45 mg/kg/day group, perhaps secondary to the lower offspring survival in that group. There were no test substance-related or statistically significant effects on sex ratio for 15 mg/kg/day and below.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL for offspring was not achieved due to decreased pup body weights at all doses and mortality at 5 and 45 (but not 15) mg/kg/day.

Remarks on result:

not determinable

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The embryofoetal and postnatal offspring toxicity at all doses was most likely caused by the profound maternal toxicity (including mortality) at all doses, accompanied by small spleens, stomach ulceration/erosion, reduced thymus weights, and associated histopathology in the spleen, thymus, kidneys, and adrenal cortex in dams at all doses. The maternal effects caused offspring in utero deaths at 45 mg/kg/day, postnatal deaths at 5 and 45 mg/kg/day, and reduced pup weights at all doses. The decreased offspring body weights at all doses and the increased pup mortalities at 5 and 45 mg/kg/day are not developmental toxicity but indicate primary effects on the maternal animals (systemic toxicity to reproductive toxicity, leading to developmental toxicity), resulting in offspring consequences. There were no reproductive or developmental toxicity NOAELs achieved in this study.