Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, aromatics (2-30 %)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

No reproductive toxicity/fertility data is available for Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, aromatics (2-30%). However, data is available for structural analogues, Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) and JP-8. Petroleum substances of similar carbon number and aromatic content, principally kerosene and jet fuel, are typically in the range of C9-C16. These substances also contain similar types of molecules in similar proportions to those in C14-C20 aliphatic [2-30% Aromatics] Hydrocarbon solvents. In general, hydrocarbon solvents are more highly refined than petroleum substances. Accordingly, the petroleum substances typically represent a “worse case” with respect to hydrocarbon solvents and can be used for read across on that basis. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%):

NOAEC for reproductive toxicity in rats ≥ 300 ppm (1720 mg/m³)

JP-8

NOAEL for reproductive toxicity in male rats ≥3000 mg/Kg/day

NOAEL for reproductive toxicity in female rats ≥1500 mg/Kg/day

Additionally, OECD 443 tests are proposed for structural analogues, Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%) (EC# 919-006-8) and Hydrocarbons, C11-C15, aromatics, <1% naphthalene (EC# 922-153-0). This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

An OECD Guideline 422 screening reproductive/developmental toxicity study (oral route) in rodents is also planned with Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, aromatics (2-30 %) (EC# 920-360-0). This endpoint will be updated subsequent to ECHA's approval of the testing proposals and availability of data upon completion of the studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Age at study initiation: males - 10 weeks, females - 9 weeks at initiation of pre-treatment mating period, 8 weeks at initiation of post-treatment mating period
- Weight at study initiation: 281-289 g
- Housing: stainless steel wire mesh cages, animals were housed individually during exposure and at a 2:1 female/male ratio during mating
- Diet (e.g. ad libitum): Purina Laboratory Chow, ad libitum
- Water (e.g. ad libitum): ad libitum

IN-LIFE DATES: From: August 21, 1978 To Oct. 13, 1978

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: one cubic meter stainless steel and glass chamber
- Method of conditioning air: The MRD-78-25 (300 ppm) was transferred from a 500 ml Erlenmeyer flask using a metering pump, or a 50 cc Tomac glass syringe into a heated flask (100 ppm) and flash evaporated. Clean air was also passed through the flask to pick up vapor. The vapor air mixture was then fed into the chamber inlets and diluted to the desired concentration. The MRD-78-26 was put in fritted bottom gas-washing bottles (400 and 1200 ppm). Air was passed through the bottles, and the vapor air mixture was then fed into the chamber inlets and diluted to the desired concentration.
- Air flow rate: 132 l/min
- Air change rate: complete air change every 7.6 min, with a 99% equilibration time of 35 min.

Details on mating procedure:

Each male cohabitated for two weeks with two females. Females were sacrificed 18 days after beginning cohabitation. Males were then exposed to the test substance for 8 weeks. Two hours after the last exposure, two untreated virgin females were placed in the males cages. These females cohabitated for seven days and replaced with two new females.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Brief description of analytical method used: IR spectrum taken with a Miran IA Ambient Air Analyzer (Wilks Scientific Corp.), analyzed at 3.4 microns.
- Samples taken from breathing zone: yes, at 1, 3, and 5 hrs after exposure began each day

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days/week for 8 weeks

Remarks:

Doses / Concentrations:
100 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
300 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

10 males, 40 females

Control animals:

yes, concurrent no treatment

Positive control:

triethylenemelamine
- Justification for choice of positive control(s): Triethylenemelamine has been shown to induce dominant-lethal mutations
- Route of administration: intraperitoneally
- Doses / concentrations: 0.5 mg/kg

Parental animals: Observations and examinations:

Animals were examined for mortality, pharmacological observations, toxicological observations (twice daily), physical observations, body weight (weekly), gross necropsy, and histopathology (seminal vesicles, epididymis, testes, prostate).

Postmortem examinations (parental animals):

The following organs were examined in males: seminal vesicles, epididymis, testes, prostate.

Statistics:

Comparisons between controls and treatment groups were made using the Chi-square method. Data was compared using the F-test and student's t-test, with the student's t-test modified using Cochran's approximation.

Reproductive indices:

Males were considered fertile if at least one female became pregnant.

Offspring viability indices:

implantation sites, early resorption sites, late resorption sites, viable fetal swellings

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

There was no mortality during the study. No treatment related physical observations were noted. Body weights were comparable between exposed animals and controls, except for the 100 ppm exposure group. However, this difference was not statistically significant. No statistically significant difference in pregnancy rates was noted. Statistically significant differences in the number of corpora lutea were noted, however, since females were not treated this was not considered treatment related. The implantation efficiency of the 100 ppm group at week 2 was significantly decreased as compared to the negative controls. This effect was deemed not to be treatment related as the effect was not seen in the 300 ppm dose group. The gross necropsy findings were unremarkable, as were the histopathological examinations.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 300 ppm

Sex:

male/female

Basis for effect level:

other: 1720 mg/m3

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

The implantation efficiency of the 100 ppm group at week 2 was decreased as compared to the negative controls. However, as the implantation efficiency was within the range of values noted for the pre-treatment mating, this was not considered to be treatment related. The mean number of early fetal deaths was comparable to negative control values. Late fetal deaths could not be evaluated, as there was an insufficient number. Total number of fetal deaths were comparable between treatment and negative controls. The gross necropsy findings were unremarkable, as were the histopathological examinations.

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

>= 300 ppm

Sex:

male/female

Basis for effect level:

other: 1720 mg/m3

Reproductive effects observed:

not specified

Mean Body Weights (g)

Week	Negative Control	Positive Control	100 ppm	300 ppm
Initial	289	281	283	284
Pre-treatment mating 1	331	325	328	328
Pre-treatment mating 2	365	362	367	363
Treatment 1	413	418	416	407
Treatment 2	438	449	441	428
Treatment 3	452	470	458	443
Treatment 4	470	487	475	459
Treatment 5	480	504	490	470
Treatment 6	484	516	502	479
Treatment 7	496	531	518	487
Treatment 8	504	538	527	500
Post-treatment mating 1	514	508	535	507
Post-treatment mating 2	523	518	544	516

Reproduction Data

Pregnancy Rate (%)
Week	Negative Control	Positive Control	100 ppm	300 ppm
Pre-treatment mating 1	75.0	70.0	70.0	70.0
Pre-treatment mating 2	80.0	85.0	90.0	95.0
Post-treatment mating 1	85.0	75.0	80.0	65.0
Post-treatment mating 2	100.0	80.0	95.0	100.0
Mean Corpora Lutea
Pre-treatment mating 1	12.5	11.8	12.9	14.0
Pre-treatment mating 2	14.1	14.9	13.1	13.5
Post-treatment mating 1	13.1	11.1	13.9	13.4
Post-treatment mating 2	13.4	11.9	14.4	13.1
Mean Implantations
Pre-treatment mating 1	10.1	11.4	12.0	13.0
Pre-treatment mating 2	12.5	14.0	12.1	11.9
Post-treatment mating 1	11.9	8.8	12.6	12.6
Post-treatment mating 2	12.7	4.1	12.8	12.5
Implantation Efficiency
Pre-treatment mating 1	80.9	96.4	92.8	92.9
Pre-treatment mating 2	88.5	94.1	92.3	88.3
Post-treatment mating 1	91.0	79.0	91.0	94.3
Post-treatment mating 2	95.1	34.0	89.4	95.4
Mean Early Fetal Death
Pre-treatment mating 1	0.2	0.4	0.6	0.5
Pre-treatment mating 2	0.6	0.5	0.5	1.0
Post-treatment mating 1	0.8	5.9	0.5	0.8
Post-treatment mating 2	0.5	4.1	0.9	0.5
Mean Late Fetal Death
Pre-treatment mating 1	0.1	0.0	0.0	0.0
Pre-treatment mating 2	0.0	0.0	0.0	0.1
Post-treatment mating 1	0.0	0.1	0.0	0.0
Post-treatment mating 2	0.0	0.0	0.0	0.0
Viable Fetal Swellings
Pre-treatment mating 1	9.9	10.9	11.4	12.5
Pre-treatment mating 2	11.9	13.5	11.6	10.8
Post-treatment mating 1	11.1	2.8	12.1	11.8
Post-treatment mating 2	12.2	0.0	11.9	12.1

Conclusions:

The NOAEC for reproductive and developmental screening is 300 ppm in rats via inhalation.

Executive summary:

This study was conducted to assess the reproductive and developmental toxicity potential of MRD-78-25 when administered to male rats. Male rats were cohabitated for two weeks with two female rats. Males were exposed for 6 hrs/day, 5 days/week, for 8 weeks. At the end of the 8 week exposure period, the male rats the cohabitated for 7 days with two virgin female rats. After this cohabitation, the males were again cohabitated with two new virgin females for another 7 days. 18 days after the beginning of cohabitation, the females were sacrificed. There were also a negative control group, and a positive control group exposed to triethylenemelamine prior to mating. Animals were examined for mortality, pharmacological observations, toxicological observations, physical observations, body weight, gross necropsy, and histopathology. Males proven fertile were then exposed to 100 or 300 ppm of test substance vapors via inhalation (10 males per concentration). The number of implantation sites, early resorption sites, late resorption sites, and viable fetal swellings were also examined. Pregnancy rates, implantation rate, and implantation efficiency were comparable between exposure groups and negative controls. The NOAEC for reproductive screening is 300 ppm for rats via inhalation.

Reference 2

Endpoint:

one-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented study report which meets basic scientific principles.

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

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:

Male rats were given 0, 750, 1500 or 3000 mg/kg neat JP-8 daily by gavage for 70 days prior to mating with naive females to assess fertility and sperm parameters.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Labs, Kingston, NY
- Weight at study initiation: (P) Males: 180 to 220 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

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:

In order to stagger delivery dates, male rats were paired with more than one female rat between 70 and 90 days of dosing with JP-8. Male rats were gavaged during cohabitation and returned to individual cages after successful mating. Exposure was continued until the rats were euthanized by carbon dioxide overdose at 90 days

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily; 7 days/ week

Details on study schedule:

The rats were given 0 (control), 750, 1500 or 3000 mg/kg JP-8 daily by gavage for 70 days prior to mating with naive females. Male rats were cohabitated with one female at a time. In order to stagger delivery dates, male rats were paired with more than one female rat between 70 and 90 days of dosing with JP-8. Male rats were gavaged during cohabitation and returned to individual cages after successful mating.

Remarks:

Doses / Concentrations:
0 (control), 750, 1500 or 3000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

20 males

Control animals:

yes, sham-exposed

Positive control:

none

Parental animals: Observations and examinations:

body weight and mortality

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

The epididymides were collected from each male rat at necropsy. The epididymides were then minced in phosphate buffered saline with bovine serum albumin; the resulting sperm suspension was videotaped in a Petroff Hauserr chamber. Determinations were made from the videotape using the CellSoft Automated Semen Analyzer (CRYO Resources, Ltd., New York, NY). Motility parameters measured by the CellSoft Analyzer were: sperm concentration, motile sperm concentration, percent motility, velocity, linearity, maximum amplitude of lateral head displacement (ALH), mean ALH and beat/cross frequency. The CellSoft Analyzer also measured the following parameters: mean radius, number of circular cells, percent circular cells/motile cells and percent circular cells/all cells.

Litter observations:

not examined

Postmortem examinations (parental animals):

not examined

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:

Unexposed females mated with dosed males were allowed to give birth in order to determine gestation length. Successful mating (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. All rats were euthanized by carbon dioxide overdose.

Offspring viability indices:

not examined

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:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

There were no clinical signs of toxicity other than changes in body weight. Body weights in male rats decreased in a dose dependent manner during 90 days gavage exposure to JP-8. A delay in starting dosing after randomly assigning rats to groups resulted in the 3000 mg/kg/day dose group being significantly heavier during the first weeks (p<0.05). Body weights were not different between groups from days 9 through 25 of exposure. From day 26 through the end of the study, the 3000 mg/kg/day dose group was significantly lighter than the control group (p<0.05). Mortality was limited to one male rat in the 750 mg/kg/day dose group.

Gestation parameters for unexposed females mated with treated males are shown below (Table 1). Pregnancy rates and gestation lengths were not adversely affected by paternal gavage exposure to JP-8. These parameters were not significantly different between dose groups. As a whole, these dams had low pregnancy rates, including the controls.

Epididymal sperm samples from males exposed by gavage to 0, 750, 1500 and 3000 mg/kg/day JP-8 for 90 days were evaluated using the CellSoft Automated Semen Analyzer. Table 2 contains sperm values for each dose group. The number of male rats per group ranged from 20 to 23. Outliers were removed after rigorous statistical analysis and were not related to dose. Significant differences were not found under any condition of analysis.

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 3 000 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: Lack of adverse treatment-related effects observed at the highest dose tested.

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Remarks on result:

not measured/tested

Remarks:

F1 parameters were not examined in the study

Reproductive effects observed:

not specified

TABLE 1. GESTATION PARAMETERS OF UNEXPOSED DAMS MATED TO MALE RATS

Dose Group	Number of Dams	Pregnancy Rate	Gestation Length
mg/kg/day	(n)	(%)	mean (+/- SE)
0	36	47	21.24 (0.26)
750	38	39	21.07 (0.18)
1500	42	57	21.08 (0.15)
3000	32	53	21.41 (0.12)

TABLE 2. SPERM PARAMETERS (MEAN ± SE) IN MALE RATS EXPOSED

Parameter	0 mg/kg/day	750 mg/kg/day	1500 mg/kg/day	3000 mg/kg/day
	(n=21)	(n=21)	(n=23)	(n=20)
Percent Motile	25.06 +/- 2.07	29.60 +/- 2.26	25.10 +/- 1.59	24.60 +/- 2.01
Conc. Motile (million/mL)	0.21 +/- 0.02	0.19+/- 0.02	0.20 +/- 0.02	0.16 +/- 0.02
Mean Velocity (um/s)	112.21 +/- 4.14	122.59 +/- 5.64	117.85 +/- 5.09	117.04 +/- 4.84
Mean Linearity	3.74 +/- 0.14	3.70 +/- 0.21	4.27 +/- 0.16	4.04 +/- 0.21
Max ALH (um)	4.04 +/- 0.19	4.28 +/- 0.24	4.06 +/- 0.15	4.00 +/- 0.19
Mean ALH (um)	3.44 +/- 0.15	3.64 +/- 0.18	3.47+ 0.1	3.41 +/- 0.14
Beat/Cross Frequency Hz (1/s)	10.42 +/- 0.28	10.34 +/- 0.23	10.86 +/- 0.19	10.70 +/- 0.27
Avg Radius (um)	16.04 +/- 1.19	15.67 +/- 0.92	15.93 +/- 1.22	13.79 +/- 0.93
Circular % of Motile	14.18 +/- 1.43	13.96 +/- 1.72	17.26 +/- 1.86	16.38 +/- 2.3
Circular % of All Cells	3.87 +/- 0.63	4.33 +/- 0.71	4.23 +/- 0.5	4.30 +/- 0.72

Conclusions:

The NOAEL ≥3000 mg/kg/day for male rat fertility.

Executive summary:

Male rats were given 0, 750, 1500 or 3000 mg/kg neat JP-8 daily by gavage for 70 days prior to mating with naive females to assess fertility and sperm parameters. Males were allowed to mate while continuing to receive treatment. Aside from a decrement in male body weight, no clinical signs were observed. There were no statistical differences noted in any reproductive parameter measured. The reproductive NOAEL ≥3000 mg/kg/day for male rats.

Reference 3

Endpoint:

one-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented study report which meets basic scientific principles.

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

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

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

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.

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

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.

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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).

Key result

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

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

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.

Key result

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

Reproductive effects observed:

not specified

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.

Reference 4

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

The 'Justification for the read across' is provided in the 'Attached justification' section below.

Species:

rat

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

2 weight of evidence read across studies from a structural analogue available for assessment.

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 720 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

One Guideline OECD 421 reproductive toxicity screening study available from structural analogues.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No reproductive toxicity/fertility data is available for Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, aromatics (2-30%). However, data is available for structural analogues, Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) and JP-8. Petroleum substances of similar carbon number and aromatic content, principally kerosene and jet fuel, are typically in the range of C9-C16. These substances also contain similar types of molecules in similar proportions to those in C14-C20 aliphatic [2-30% Aromatics] Hydrocarbon solvents. In general, hydrocarbon solvents are more highly refined than petroleum substances. Accordingly, the petroleum substances typically represent a “worse case” with respect to hydrocarbon solvents and can be used for read across on that basis. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)

A key read across OECD Guideline 421 screening reproductive toxicity study (ExxonMobil, 1980a), was conducted to assess the reproductive and developmental toxicity potential of the test material (Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)) when administered to male rats. Male rats were cohabitated for two weeks with two female rats. Males were exposed for 6 hrs/day, 5 days/week, for 8 weeks. At the end of the 8 week exposure period, the male rats the cohabitated for 7 days with two virgin female rats. After this cohabitation, the males were again cohabitated with two new virgin females for another 7 days. 18 days after the beginning of cohabitation, the females were sacrificed. There were also a negative control group, and a positive control group exposed to triethylenemelamine prior to mating. Animals were examined for mortality, pharmacological observations, toxicological observations, physical observations, body weight, gross necropsy, and histopathology. Males proven fertile were then exposed to 100 or 300 ppm of test substance vapors via inhalation (10 males per concentration). The number of implantation sites, early resorption sites, late resorption sites, and viable fetal swellings were also examined. Pregnancy rates, implantation rate, and implantation efficiency were comparable between exposure groups and negative controls. The NOAEC for reproductive screening was determined to be ≥300 ppm for rats via inhalation.

JP-8

In a read across study (Mattie et al., 2000), male rats were given 0, 750, 1500 or 3000 mg/Kg neat JP-8 daily by gavage for 70 days prior to mating with naive females to assess fertility and sperm parameters. Males were allowed to mate while continuing to receive treatment. Aside from a decrement in male body weight, no clinical signs were observed. There were no statistical differences noted in any reproductive parameter measured. The reproductive NOAEL was ≥3000 mg/Kg/day for male rats.

 

In another read across study (Mattie et al., 2000), 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 was ≥1500 mg/Kg/day for female fertility. The NOAEL was ≥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.

Effects on developmental toxicity

Description of key information

No developmental toxicity data is available for Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, aromatics (2-30%). However, data is available for structural analogues, Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) and JP-8/Kerosene. Petroleum substances of similar carbon number and aromatic content, principally kerosene and jet fuel, are typically in the range of C9-C16. These substances also contain similar types of molecules in similar proportions to those in C14-C20 aliphatic [2-30% Aromatics] Hydrocarbon solvents. In general, hydrocarbon solvents are more highly refined than petroleum substances. Accordingly, the petroleum substances typically represent a “worse case” with respect to hydrocarbon solvents and can be used for read across on that basis. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)

NOAEC for developmental toxicity: ≥ 300 ppm (1720 mg/m³)

JP-8/Kerosene

NOAEL for developmental toxicity: 500 mg/Kg bw/day

Additional OECD Guideline 414 rodent and non-rodent species tests are proposed for structural analogues Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%) (EC# 919-006-8) and Hydrocarbons, C11-C15, aromatic, <1% naphthalene (EC# 922-153-0).

This endpoint will be updated subsequent to ECHA's approval of the testing proposals and availability of data upon completion of the studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

11 Sept. 1978 - 6 Oct. 1978

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: Guidelines for Reproduction Studies for Safety and Evaluation of Drugs for Human Use, Segment II (Teratology Study)

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: 7 weeks
- Fasting period before study: Animals were not given food during exposure.
- Housing: Individually, except during mating, in stainless steel cages, animals identified by ear tags
- Diet (e.g. ad libitum): Purina Lab Chow, ad libitum
- Water (e.g. ad libitum): Elizabethtown Water Company, ad libitum
- Acclimation period: Aug. 17, 1978-Sept. 4, 1978


ENVIRONMENTAL CONDITIONS
- Temperature (°C): monitored twice daily, room temperature
- Humidity (%): dry air
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark


IN-LIFE DATES: days 6-15 of gestation From: 11-27 Sept. 1978 To: 20 Sept. - 6 Oct. 1978

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: one cubic meter exposure chamber
- Temperature, humidity, pressure in air chamber: room temperature, dry air

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: Overnight and removed in morning to check for pregnancy, this was repeated until females were pregnant
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

6 hrs/day

Frequency of treatment:

days 6-15 of gestation

Duration of test:

days 6-15 of gestation

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, toxicological signs, pharmacological effects

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6-15, 21 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: 21
- Organs examined: uterus, ovaries

Ovaries and uterine content:

The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of live fetuses, number of dead fetuses

Fetal examinations:

- External examinations: Yes: all per litter examined for sex, crown-rump distance, weighed, and malformations
- Soft tissue examinations: Yes: 2/3 per litter examined for gross dissection and examination of viscera, internal sex determination, ureter, kidneys, and heart
- Skeletal examinations: Yes: 2/3 per litter examined for skeletal malformations, and ossification
- Head examinations: Yes: 1/3 per litter examined for neural defects

Statistics:

Analysis was done using the chi-square method, or the F-test and Student's t-test. When the variance differed significantly, the Student's t-test was modified suing Chochran's approximation. The mean number of live fetuses, resorptions, implantations, and corpora lutea were analyzed using the one-tailed t-test.

Indices:

implantation efficiency,

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There was no mortality in either of the dosage groups. Pregnancy rates were comparable between the exposure groups and negative controls. Weight gain was significantly higher in the exposure groups post-dosing. There were no significant clinical observations in either exposure group. The mean number of corpora lutea was significantly decreased in the 300 ppm group. Since ovulation occurred prior to exposure, this was not considered to be treatment related. The mean number of implantations was comparable between exposure groups and negative controls. The implantation efficiency values were actually significantly higher in exposure groups as compared to negative controls. The mean number of live fetuses, resorption sites, and number of dams with more than one resorption site were comparable between exposures and negative controls. The gross postmortem examination of dams showed no treatment related effects.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 300 ppm

Basis for effect level:

other: Systemic toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The body weights of fetal males in the 100 ppm group were significantly higher than negative controls. There were some statistically significant differences in mean crown-rump distance between both dosage levels and negative controls, these differences were slight and the effect inconsistant between dosages and sex. These differences were therefore not considered to be indicative of a treatment related effect. Mean numbers of male and female fetuses, and sex ratios were similar between exposure groups and negative controls. Ossification variations were similar in exposure groups and negative controls, as was the incidence of litters with fetuses containing ossification variations. No malformations externally or in the soft tissues were noted in the fetuses except in the positive controls. Though skeletal defects were noted in the exposure group, the types of malformations are common in rat fetus and not considered to be treatment related.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 300 ppm

Based on:

test mat.

Basis for effect level:

other: Developmental Toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Results - Dams

Endpoint	Negative Control	400 mg/kg ASA	100 ppm	300 ppm
Pregnancy Rate (%)	100.0	95.0	100.0	90.0
Mortality Rate (%)	0.0	10.0	0.0	0.0
Mean Body Weight Gain  (g) Dams - Days 15-21	84	32	108	103
Mean Corpora Lutea	15.2	14.5	15.5	13.8
Mean No. Implantations	13.0	13.2	13.8	13.2
Implantation Efficiency (%)	85.8	91.1	88.7	95.6
Mean No. Live Fetuses	12.5	7.4	12.9	12.6
Mean No. Dead Fetuses	0.0	0.0	0.0	0.0
Mean No. Resorptions	0.6	5.8	0.9	0.7
Dams with more than one Resorption (%)	10.0	58.8	25.0	11.1

Results – Fetuses

Endpoint	Negative Control	400 mg/kg ASA	100 ppm	300 ppm
Male Mean Fetal Weight (g)	5.57	3.88	5.82	5.62
Female Mean Fetal Weight (g)	5.29	3.62	5.44	5.33
Male Mean Crown-Rump Distance (cm)	4.3	3.7	4.4	4.2
Female Mean Crown-Rump Distance (cm)	4.2	3.6	4.2	4.1
Sex Ratio (%)	91.5	98.4	88.3	105.5
Ossification Variations (%)	70.7	100.0	79.4	79.3
Litters with Ossification Variations (%)	100.0	100.0	95.0	100.0
Soft Tissue Malformations (%)	2.4	26.8	1.1	3.9
Litters with Soft Tissue Malformations (%)	10.0	54.5	5.0	16.7
Gross Evisceration Malformations (%)	5.4	3.6	1.8	4.0
Litters with Gross Evisceration Malformations	25.0	8.3	10.0	16.7
Skeletal Malformations (%)	0.0	21.4	2.9	1.3
Litters with Skeletal Malformations	0.0	66.7	15.0	11.1

Conclusions:

The NOAEC for developmental toxicity in rats is >=300 ppm (1575 mg/m3) via inhalation. The test substance is also non-teratogenic.

Executive summary:

This study determined the developmental toxicity of MRD-78 -25 in rats exposed via inhalation. Groups of 20 pregnant female rats were exposed 6 hrs/day during days 6 -15 of gestation. Test concentrations of 100 or 300 ppm test substance. In addition to a negative control group, there was also a positive control group that was exposed to acetylsalicylic acid on days 6 -15 of gestation. Dams were observed for toxicological signs and pharmacological effects. On day 21 of gestation, the animals were sacrificed, and examined for corpora lutea and uterine implantation parameters. Fetuses were examined for fetal size, sex ratio, and external, soft-tissue, and skeletal malformations. No adverse effects due to exposure to the test substance were seen in either dams or fetuses. No treatment related malformation effects were noted in the fetuses. The developmental NOAEC for rats by inhalation is >=300 ppm. The test substance is also not teratogenic.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented study report which meets basic scientific principles.

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Time-mated Sprague-Dawley rats were dosed orally with JP-8 at 0, 500, 1000, 1500 and 2000 mg/kg/day on days 6-15 of pregnancy.

GLP compliance:

not specified

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: One hundred and fifty time-mated virus antibody-free rats were purchased on day 4 of pregnancy.
- Weight at study initiation: all groups contained dams of approximately equivalent body weight at the beginning of the study
- Diet (e.g. ad libitum): Purina Lab Chow, ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 71-73°F
- Humidity (%): 45-55% relative humidity
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

On days 6-15 of pregnancy the animals assigned to each treatment group were administered one of the following doses of JP-8 by oral gavage: 500, 1000, 1500 and 2000mg/kg/day. The volume of JP-8 administered ranged from 1.1 to 7.3 ml daily, depending on the dam's body weight and dose group. Control animals received 1 ml of sterile water daily in order to emulate any stress that may have been associated with the dosing process. Animals were examined daily for evidence of toxicity. On day 20 of pregnancy all animals were euthanized with carbon dioxide.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

GD 6-15

Frequency of treatment:

daily

Duration of test:

GD 6-15

Remarks:

Doses / Concentrations:
0, 500, 1000, 1500 and 2000mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

30 females

Control animals:

yes, sham-exposed

Maternal examinations:

Animals were examined daily for evidence of toxicity.

Ovaries and uterine content:

The dam's body weight and the liver, kidney and gravid uterus weights were recorded. The total number of corpora lutea, live fetuses, dead fetuses and resorption sites were also recorded for each dam. Uteri that appeared to be non-gravid were placed in a 10% ammonium sulfate solution for 10 min and then reevaluated for the presence of early resorptions.

Fetal examinations:

Viable fetuses were sexed, weighed and examined for gross abnormalities. One-half of the fetuses were evaluations using the technique of Wilson. The remaining fetuses were placed in ethanol for later examination of skeletal anomalies using the procedure of Staples and Schnell.

Statistics:

Maternal body weight and average pup weights per litter were first analyzed using Bartlett's test for homogeneity of variance followed by one-way analysis of variance (ANOVA) procedures. Bonferoni's test was used to compare the body weights of maternal and fetal control animals with those of animals in the treatment groups. Non-parametric data (i.e. corpora lutea counts, number of live fetuses and the number of dead fetuses) were analyzed using the Kruskal- Wallis test followed by the Mann-Whitney U test when appropriate. The incidence of fetal malformations/variations per litter were compared using Fisher's exact test. In all cases P <= 0.05 was used as the level of significance.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Significant maternal toxicity was evidenced by a reduction in maternal body weight gain in the 1000, 1500 and 2000 mg/kg/day dose groups. Maternal necropsy weight (maternal body weight minus pregnant uterus's weight) was also significantly different from that of the control animals at doses of 1500 and 2000 mg/kg/day. No clinically apparent maternal toxicity was noted during the course of the study. Thirteen animals were found dead in their cage at various times during the dosing period and each was submitted to necropsy. In all cases the cause of death was found to be related to the presence of JP-8 in the lungs. Maternal observations at necropsy, including the number of females pregnant, number of corpora lutea per female, number of fetuses per female and post-implantation loss, were within the normal range for all treatment groups.

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The average body weight of male and female fetuses was significantly reduced in the 1500 and 2000 mg/kg/day dose groups. A detailed examination of the fetal tissues revealed no significant increase in the incidence of malformations or variations in treated versus control animals. The sex ratio of male and female fetuses was also similar among the control and dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: fetotoxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The maternal NOAEL was determined to be 500 mg/kg/day based on reduced maternal body weight. The fetotoxicity NOAEL was determined to be 1000 mg/kg/day based on reduced fetal weight. There were no developmental or teratogenic effects noted (aside from body weight reduction) in any dose tested.

Executive summary:

Time-mated Sprague-Dawley rats were dosed orally with JP-8 at 0, 500, 1000, 1500 and 2000 mg/kg/day on days 6-15 of pregnancy. The maternal NOAEL was determined to be 500 mg/kg/day based on reduced maternal body weight. The fetotoxicity NOAEL was determined to be 1000 mg/kg/day based on reduced fetal weight. There were no developmental or teratogenic effects noted (aside from body weight reduction) in any dose tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

One read across oral developmental toxicity study from a structural analogue available for assessment.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 575 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Two inhalation developmental toxicity studies from structural analogues available for assessment.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No developmental toxicity data is available for Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, aromatics (2-30%). However, data is available for structural analogues, Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) and JP-8/Kerosene. Petroleum substances of similar carbon number and aromatic content, principally kerosene and jet fuel, are typically in the range of C9 -C16. These substances also contain similar types of molecules in similar proportions to those in C14-C20 aliphatic [2-30% Aromatics] Hydrocarbon solvents. In general, hydrocarbon solvents are more highly refined than petroleum substances. Accordingly, the petroleum substances typically represent a “worse case” with respect to hydrocarbon solvents and can be used for read across on that basis. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)

A key study (ExxonMobil, 1979b) determined the developmental toxicity of the test material (Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)) in rats exposed via inhalation. Groups of 20 pregnant female rats were exposed 6 hrs/day during days 6 -15 of gestation. Test concentrations of 100 or 300 ppm test substance. In addition to a negative control group, there was also a positive control group that was exposed to acetylsalicylic acid on days 6 -15 of gestation. Dams were observed for toxicological signs and pharmacological effects. On day 21 of gestation, the animals were sacrificed, and examined for corpora lutea and uterine implantation parameters. Fetuses were examined for fetal size, sex ratio, and external, soft-tissue, and skeletal malformations. No adverse effects due to exposure to the test substance were seen in either dams or fetuses. No treatment related malformation effects were noted in the fetuses. The developmental NOAEC for rats by inhalation was determined to be >=300 ppm. The test substance is also not teratogenic.

JP-8/Kerosene

In a study (Cooper and Mattie, 1996), time-mated Sprague-Dawley rats were dosed orally with JP-8 at 0, 500, 1000, 1500 and 2000 mg/Kg/day on days 6-15 of pregnancy. The maternal NOAEL was determined to be 500 mg/Kg/day based on reduced maternal body weight. The fetotoxicity NOAEL was determined to be 1000 mg/Kg/day based on reduced fetal weight. There were no developmental or teratogenic effects noted (aside from body weight reduction) in any dose tested.

Justification for classification or non-classification

Based on available read across data from structural analogues, Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, aromatics (2-30%) does not warrant the classification as a reproductive or developmental toxicant under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP). However, further tests (OECD 422, 443, and OECD 414 (rodent and 2nd species)) are proposed on the substance and structual analogues, respectively, and will be conducted subsequent to ECHA's approval of the same. This endpoint will be updated upon completion of the above studies subject to ECHA's approval.