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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
Female rats were dosed with neat JP-8 (0, 325, 750, 1500 mg/kg) daily by gavage for a total of 21 weeks (90-day plus mating with naive males, gestation and lactation) in an effort to assess general toxicity, fertility and reproductive endpoints.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
The JP-8 jet fuel was supplied by the U.S. Air Force (AFRL Propulsion Directorate, Wright- Patterson AFB, OH). The fuel met the requirements of Military Specification MIL-T-83133A. JP-8 was administered by gavage without a vehicle (neat). Control animals were dosed with 1.0 mL distilled water under the same conditions as test groups. Volumes to be administered each day were calculated from the individual daily body weights and the density of the test material (0.81 g/mL).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Labs, Kingston, NY
- Weight at study initiation: (P) Females: 180 to 200 g
- Diet (e.g. ad libitum): Formula 5008, Ralston Purina, St. Louis, MO, ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
JP-8 was administered by gavage without a vehicle (neat). Control animals were dosed with 1.0 mL distilled water under the same conditions as test groups. Volumes to be administered each day were calculated from the individual daily body weights and the density of the test material (0.81 g/mL).
Details on mating procedure:
The rats were given 0 (control), 325, 750 or 1500 mg/kg JP-8 daily by gavage for 21 weeks (90-days followed by cohabitation, gestation, delivery and lactation). The male rats, not exposed to JP-8, were housed 1:1 with treated female rats. Dams were euthanized one day after weaning (Day 22 of lactation); male rats were euthanized after pregnancy was confirmed. Litters were standardized to four male pups and four female pups on postnatal day (PND) 5. All rats were euthanized by carbon dioxide overdose.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days followed by cohabitation, gestation, delivery and lactation
Frequency of treatment:
daily; 7 days/ week
Details on study schedule:
Young female Sprague-Dawley rats weighing 180-200 g were randomly assigned to 4 exposure groups. Groups contained a minimum of 35 female rats. The rats were given 0 (control), 325, 750 or 1500 mg/kg JP-8 daily by gavage for 21 weeks (90-days followed by cohabitation, gestation, delivery and lactation). The male rats, not exposed to JP-8, were housed 1:1 with treated female rats. Dams were euthanized one day after weaning (Day 22 of lactation); male rats were euthanized after pregnancy was confirmed. Litters were standardized to four male pups and four female pups on postnatal day (PND) 5. All rats were euthanized by carbon dioxide overdose.
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 325, 750 or 1500 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
35 females
Control animals:
yes, sham-exposed
Positive control:
none

Examinations

Parental animals: Observations and examinations:
A subset of dams from each treatment group (maximum n of 10) was selected for hematology, clinical chemistry and urine analyses. The same subsets were also used for organ weights and histopathology. Whole blood was collected from the dam's inferior vena cava at necropsy. The following hematology parameters were measured: red blood cell count, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, red cell distribution width, mean corpuscular hemoglobin concentration, hematocrit, platelet count and differential leukocyte count. Determinations were made using an automated counter (H-1 System, Technicon Instruments, Corporation, Tarrytown, NY).

The following clinical chemistry parameters were measured in serum from dams: sodium,
glucose, magnesium, carbon dioxide, potassium, albumin, chloride, total protein, calcium, blood urea nitrogen, total bilirubin, uric acid, inorganic phosphate, creatinine, triglycerides, cholesterol, AST, ALT, alkaline phosphatase, lactate dehydrogenase, creatine kinase and gamma-glutamyl transferase. Assays were performed with the Ektachem 700XR chemistry analyzer (Eastman Kodak, Rochester, NY).

The dams were transferred to metabolism cages upon weaning and urine was collected for 24- hours prior to sacrifice. The total volume of urine was noted and the urine was assayed for pH, specific gravity, total protein and creatinine. Specific gravity was measured with a refractometer (American Optical Corp., Southbridge, MA) and pH was measured with an electronic meter (Model 601A, Orion Research Inc., Cambridge, MA). Urine protein assays were performed on an automated chemistry analyzer (Model ACA IV, DuPont, Wilmington, DE). Urine creatinine determinations were made using the Ektachem 700XR analyzer.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
not examined
Postmortem examinations (parental animals):
A gross pathologic examination was performed on a subset of dams from each treatment group following euthanasia. A maximum of 10 rats per group was designated for clinical pathology and histopathology. Tissues were collected and prepared for histopathologic examination and included: gross lesions, thymus, brain, kidneys, lungs, adrenals, trachea, pancreas, heart, ovaries, uterus, liver, nasal turbinates, spleen, esophagus, duodenum, stomach, jejunum, colon, ileum, rectum, urinary bladder, sternum, mandibular lymph nodes, sciatic nerve, mesenteric lymph nodes, skeletal muscle. Brain, kidneys, liver, spleen and ovaries were weighed during necropsy.
Postmortem examinations (offspring):
not examined
Statistics:
Statistical Analyses for General Toxicity Data
Adult body weights, organ weights and urine metabolites were analyzed by an ANOVA with multiple comparisons. Clinical chemistry results, hematology values, urinalysis data and severity of pathological changes were compared using an ANOVA. The level of significance was accepted at p<0.05 unless stated otherwise.

Statistical Analyses for Reproductive Measures
A one-factor (dose) or two-factor (dose and pup sex) analysis of variance (ANOVA) was performed for continuous variables. Error terms used were either dam(dose) or pup sex and dam(dose). One-way ANOVA was used with gestation lengths, sperm parameters and litter sizes while two-way was used for pup weights. Post-hoc paired comparisons of dose used two-tailed t-tests with pooled error.

For categorical variables, a Chi-square test of proportions was used to determine differences among the doses. Chi-square tests were used for pregnancy rates and percent viability. Posthoc paired comparison for the viability parameter was performed with Fisher's Exact test. The level of significance was accepted at p<0.05 unless stated otherwise.
Reproductive indices:
Gestation day 0 was determined by presence of copulatory plug or sperm in a vaginal contents smear. Pregnancy rate (%) and gestation duration (days) were recorded for all dams. Size of the entire litter and the number born dead were noted on PND 1; the resulting numbers were compared between treatment groups.
Offspring viability indices:
Size of the entire litter and the number born dead were noted on PND 1; the resulting numbers were compared between treatment groups. Pups were weighed on PND 1, 4, 7, 14, 21 and 90; male and female pup weights were compared between treatment groups and between sexes.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
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)

Results of the 21-week oral gavage exposure to 0, 325, 750 and 1500 mg/kg/day JP-8 revealed a decrease in body weights of the female rats. Body weights for the 1500 mg/kg/day rats were significantly lower than control rats (p<0.01) starting at week 8 and continuing throughout gestation and most of lactation (weeks 13-20). Terminal body weights at week 21 were not significantly different from control rat weights. Mortality in each treatment group was not related to dose.

Pregnancy rates and litter sizes for treated animals were not significantly different from controls. Gestation length was calculated from dams that became pregnant within one estrous cycle. Of the 87 dams that became pregnant, 77 took 1 to 4 days of cohabitation to become pregnant. The remaining 13 dams had reported times to impregnation ranging from 5 to 11 days. Most of these dams had gestation lengths as short as 14 days due to misidentification of the first day of impregnation. Therefore, time to impregnation plus gestation length was determined for each group. Since there were no significant differences between control and exposure groups for the combined impregnation/gestation length, dams with long impregnation times and short gestation lengths were excluded from the gestation length calculation. There were still no significant differences seen in gestation length for the remaining dams. The percentage of live pups on Day 1 for each dose group was not different from the control percent. The number of dams per treatment with at least one dead pup was not different between the dose groups and control.

No significant changes were found in urine parameters (total volume, specific gravity and creatinine concentration). There were no statistically significant changes in most hematology counts: neutrophil, eosinophil, basophil, lymphocyte and platelet. The leukocyte count was found to be significantly decreased in the 325 mg/kg/day dose group alone (p<0.05). This change was not dose dependent and has questionable biological significance. Clinical chemistry values (sodium, chloride, glucose, triglycerides, creatinine, alkaline phosphatase, AST, ALT) for treatment groups were not significantly different from control values. Data are not shown for urine, hematology and clinical chemistry parameters.

Samples of all collected tissues for each rat in the subsets were not available for histopathological examination. Significant pathological changes were limited to squamous hyperplasia of the stomach and perianal dermatitis. The incidence and severity of these changes were found to be dose-dependent and statistically significant at 1500 mg/kg/day for perianal dermatitis and stomach hyperplasia (p<0.05, see Table 6). Only the incidence and severity of the squamous hyperplasia of the stomach were significantly increased after oral exposure to 750 mg/kg/day JP-8 (p<0.05).

One day after weaning, the dams were euthanized. A subset from each group was necropsied. Due to sacrifices falling on weekends and the deaths of three animals not related to JP-8 dose (two from the 325 and one from the 750 mg/kg/day groups), the number of female rats per subset was limited. Treatment subsets had 7 or 8 rats while the control subset had 10 rats available. Liver weights were significantly increased, as were liver to body weight and liver to brain weight ratios (p<0.01 in 1500 mg/kg/day group). Kidneys to brain ratios were also significantly increased (p<0.05).

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 1 500 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

There was no difference in survival of pups between control and dose groups at PND 4, 14 and 21. Three pups, each from different litters in the 750 mg/kg/day dose group, did not survive between PND 21 and PND 90. Only one pup from the 1500 mg/kg/day group died during this time.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
750 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: based on decreased pup weight

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL >=1500 mg/kg/day for female fertility.
Executive summary:

Female rats were dosed with neat JP-8 (0, 325, 750, 1500 mg/kg) daily by gavage for a total of 21 weeks (90-day plus mating with naive males, gestation and lactation) in an effort to assess general toxicity, fertility and reproductive endpoints. The NOAEL >=1500 mg/kg/day for female fertility. The NOAEL >=1500 mg/kg/day for female fertility. The NOAEL for the pup was 750 mg/kg/day based on a decrement in body weight which correlated with a decrease in maternal body weight at 1500 mg/kg/day.

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