Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive Toxicity

READ ACROSS DATA: C16-C30 highly purified light mineral oil (60% paraffins, 40% naphthenes)

1-generation reproductive/developmental Test (OECD TG 415) - Dermal administration in females.

NOAEL for fertility, maternal toxicity and developmental toxicity >= 2000 mg/kg bw/day (highest dose tested)

READ ACROSS DATA: C16-C30 highly purified light mineral oil (60% paraffins, 40% naphthenes)

1-generation reproductive/developmental Test (OECD TG 415) - Dermal administration in males; 13-weeks prior to mating.

NOAEL for male fertility (sperm count, sperm morphology and spermatogenesis) for mineral oil >= 2000 mg/kg bw/day (highest dose tested)

READ ACROSS DATA: C13-C22 hydrocarbon fuel (30% n- & 70% iso-paraffins)

2-generation reproductive/developmental Test (OECD TG 416) - Oral administration in males and females.

NOAEL for fertility and developmental toxicity >= 750 mg/kg bw/day

SUPPORTING DATA:  JP-8 Fuel (C9-C16 Aliphatics, 25% aromatics)

1-Generation Reproduction Toxicity Study (OECD TG 415) - Male Fertility Test – Oral Administration - 90d prior to mating, the NOAEL >=3000 mg/kg/day, which was the highest dose tested.

Female fertility NOAEL >= 1500 mg/kg (highest dose tested). Developmental NOAEL = 750 mg/kg (decreased BW).

READ ACROSS DATA: C9-14 aliphatics (<2% aromatic)

 

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD TG 422) – Oral Administration (decane) - The NOAEL for developmental toxicity was ≥1000 mg/kg/day and the NOAEL for reproductive toxicity was ≥1000 mg/kg/day.  

 

Reproduction / Developmental Toxicity Screening Test (OECD TG 422) - Oral Administration (undecane) - The NOAEL for developmental toxicity was ≥1000 mg/kg/day and the NOAEL for reproductive toxicity was ≥1000 mg/kg/day.

 

READ ACROSS DATA: C9-14 aliphatics (2-25% aromatic)

Reproduction / Developmental Toxicity Screening Test (OECD TG 421) - Inhalation Administration - The NOAEL for developmental toxicity was ≥300 ppm (1720 mg/m3).

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: F0 animals were approximately 6 to 7 weeks (42 to 46 days) of age at the initiation of test item
administration.
- Weight at study initiation: 142.4 to 190.6 g; female group mean range 137.4 to 165.1 g.
- Fasting period before study: Not applicable.
- Housing: During the acclimation period (F0 animals only) and throughout the study, all F0 and F1 parental animals were housed
individually, except during the mating period, in solid-bottom polycarbonate cages with stainless steel wire lids. The rats were paired
for mating in the home cage of the male. Following positive evidence of mating (or at the completion of the 2-week mating period),
the females were transferred to individual solid-bottom polycarbonate cages with stainless steel wire lids. Beginning on pre-natal
Day 21, the selected F1 pups were individually housed until the start of the mating period.
- Diet: Purina Certified Pelleted Rodent Diet® (No. 5002, PMI Feeds, Inc., St. Louis, MO) was available ad libitum in the wire cage
lids. Available information on the diet did not indicate the presence of any substance at a concentration likely to have influenced the
outcome of the study.
- Water (tap water) was available ad libitum via plastic water bottles with butyl rubber stoppers and stainless steel sipper tubes.
Available information on the water did not indicate the presence of any substance at a concentration likely to have influenced the
outcome of the study.
- Acclimation period: Seven days

ENVIRONMENTAL CONDITIONS
- Temperature: (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): At least ten air changes per hour
- Photoperiod (hr dark / hrs light):12 hours continuous light and 12 hours darkness
IN-LIFE DATES: First day of treatment: 13 October 2009 - Final necropsy day was 07 July 2010.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared as solutions at concentrations of 0 (vehicle), 25, 100, and 375 mg/mL
were formulated 10 times (approximately every 4 weeks), dispensed into 30-mL amber glass bottles with Teflon-lined lids for use as
daily dose aliquots, and stored at room temperature protected from light. Concentrations of 25 and 375 mg/mL in the same vehicle
have been shown to be homogeneous and stable for 42 days when stored refrigerated or at room temperature protected from light
(Analytical Chemistry report. Report date: 10 March 2010).
The test item formulations (but not the vehicle formulation) were stirred continuously throughout dose administration.

Prior to use on study, samples (~25 mL each) for homogeneity determination were collected from the top, middle, and bottom strata
of each test item formulation from 3 of the 10 formulation dates (06 October 2009, 26 January 2010, and 15 June 2010). Samples
(~25 mL each) for concentration analysis were also collected from the middle stratum of each dosing formulation (including the
vehicle formulation) from these same 3 formulation dates. All analyses were conducted using a validated gas chromatography
method with flame ionization detection (AM-0212131-002.0). Details about the methodology and results of these analyses are
presented in Appendix 1, and summarized as follows:
Based on these results, the analyzed dosing formulations were found to contain the amount of test item prescribed in the protocol
(±10% of nominal) and were homogeneous. Therefore, the protocol-specified dosages of test item were administered to the
animals.

-DIET PREPARATION
Not applicable
- Rate of preparation of diet (frequency): Not applicable
- Mixing appropriate amounts with (Type of food): Not applicable
- Storage temperature of food: No data
- VEHICLE: Corn oil
- Justification for use and choice of vehicle (if other than water): Not applicable
- Concentration in vehicle: 0, 25, 100, and 375 mg/mL
- Amount of vehicle (if gavage): 2 ml/kg
- Lot/batch no. (if required): Not applicable
Details on mating procedure:
- M/F ratio per cage: 1/1 (Animals were paired on a 1 male: 1 female basis within each dose group).
- Length of cohabitation: Following positive evidence of mating (or at the completion of the 2-week mating period).
- Proof of pregnancy: Vaginal smears were performed daily on each F0 and F1 female for 21 days prior to pairing and continuing from the day after
pairing until evidence of mating was observed or until the end of the mating period. The slides from the 21 days prior to mating were
evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female according to test facility SOPs.
Vaginal smears were also performed on the day of necropsy to determine the stage of estrus at demise.
- After a set number of days of unsuccessful pairing replacement of first male by another male with proven fertility.: Not applicable
- Further matings after two unsuccessful attempts: Not applicable
- After mating each pregnant female was returned to individual caging: Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method has been satisfactorily validated and details about the methodology and results of these analyses are
presented in detail.
The results are summarized as follows.
Three sets of dose formulation samples were analyzed for concentration verification and homogeneity assessment for the test item
in corn oil formulations had previously been evaluated for homogeneity , in long-term storage, and animal room dosing stabilities in the concentration range from 25 to 375 mg/mL and the detailed analytical procedures were reported in an Analytical Chemistry
Report, Report Date 10 March 2010.
The formulation analyses reported here were performed following an validated analytical method to verify the test item
concentration and homogeneity in corn oil formulations prepared on October 6, 2009 and January 26 and June 15, 2010. All dose
formulations analyzed were found to be within ±10% of the nominal concentration and were homogeneous.

ANALYTICAL METHOD
The validated analytical method used to analyze study samples is outlined as follows:
For each dose formulation sample, triplicate 1 mL aliquots were in 10-mL volumetric flasks and diluted to volume with n-hexane.
Processed formulation samples were then analysed on a gas chromatograph with a flame ionization detector along with a series of
vehicle standards used to generate a calibration curve. Vehicle standards were prepared by mixing blank formulations samples.
Test item concentrations were calculated using a least square s linear regression equation that fit the relationship between the
nominal concentration s of vehicle standard and the detector response (ie the sum of integrated areas of selected peaks within the
chromatogram). The formulation sample concentrations were determined in mg/g and converted to mg/mL measuring the
formulation densities.
Duration of treatment / exposure:
Duration of treatment / exposure
All male and female for the F0 generation and F1 generation were administered the test item or vehicle control, daily for at least 70
consecutive days prior to mating.
The test item was administered to offspring selected to become the F1 parental generation following weaning, beginning on
postnatal day (PND) 22.
The F0 and F1 males continued to receive the test item throughout mating and through the day prior to euthanasia. The F0 and F1
females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia.
A complete gross necropsy was conducted after death for any parental (F0 and F1) or offspring (F1 and F2) animals found in
moribund condition or dead on study.
Organs were not weighed for moribund animals.
Histopathological examination of study specified organs were performed for all high dose and control parental (F0 and F1).
Additionally, reproductive organs of the low and mid dose animals suspected of reduced fertility were subjected to histopathological
evaluation.
For any organ(s) demonstrating possible treatment-related changes in the high dose group versus the organ(s) of interest were
evaluated histopathologically from successively lower concentration dose groups until a clear no observed adverse effect level
(NOAEL) was established. However, at the request of the Sponsor, no further evaluation of the lungs from lower dose groups was
conducted.
Frequency of treatment:
The test and vehicle control item formulations were administered to the F0 and F1 males and females and once daily for a minimum
of 70 consecutive days prior to mating and for males continued throughout mating to the day prior to termination.
The F0 and F1 females continued to be dosed throughout mating, gestation, and lactation, through to the day prior to termination.
The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) were potentially exposed to the test article in utero and
through nursing (Pre Natal Day 0 to 21).
The F1 pups selected as parents for the F2 generation (25/sex/group) were administered the test item following weaning (beginning
on Pre Natal Day 22).
Details on study schedule:
Three groups of male and female Crl:CD(SD) rats (25 and 28/sex/group for the F0 and F1 generations, respectively) were
administered the test item (CAS No. 848301-67-7), daily by oral gavage for at least 70 consecutive days prior to mating. A
concurrent control group of 25 and 28/sex for the F0 and F1 generations, respectively, received the vehicle, corn oil (CAS No.
8001-30-7) on a comparable regimen. The F0 and F1 females continued to receive the test item throughout mating, gestation, and
lactation, and through the day prior to euthanasia.
The test item was administered to offspring selected to become the F1 parental generation following weaning, beginning on
postnatal day (PND) 22. The F0 and F1 males continued to receive the test item throughout mating and through the day prior to
euthanasia.
The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) were potentially exposed to the test article in utero and
through nursing (Pre Natal Day 0 to 21). The F1 pups selected as parents for the F2 generation (25/sex/group) were administered
the test item following weaning (beginning on Pre Natal Day 22).
The first day of dosing was designated as Day 1 of the study.
Animals were paired on a 1 male: 1 female basis within each dose group.
The rats were paired for mating in the home cage of the male.
Following positive evidence of mating (or at the completion of the 2-week mating period), the females were transferred to individual
solid-bottom polycarbonate cages with stainless steel wire lids.
Remarks:
Doses / Concentrations:
50 mg/kg/day (25 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg/day (100 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg/day (mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
F0 generation
0 mg/kg/day (Control) - 25 per sex
50 mg/kg/day - 25 per sex
200 mg/kg/day - 25 per sex
750 mg/kg/day - 25 per sex

F1 generation
0 mg/kg/day (Control) - 28 per sex
50 mg/kg/day - 28 per sex
200 mg/kg/day - 28 per sex
750 mg/kg/day - 28 per sex
Details on study design:
- Dose selection rationale: Dosage levels were selected based on repeat-dose studies on similar substances and a 90-day repeat-dose study on 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' and 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and
linear'. In addition to repeat-dose studies on conventional petroleum-based diesel, a number of repeat-dose studies on individual
components of the test substance were available. These studies do not give any indication of unexpected effects and are in line
with the observations in 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' and 'Naphtha (Fischer-Tropsch),
light, C4-10 - branched and linear' studies. The selected route of administration was oral (gavage) by the Sponsor because this is a
possible route of human exposure. The number of animals selected for this study was the minimum required to yield statistically
and scientifically meaningful data and was consistent with regulatory agency expectations.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Random
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- See attached Summary Tables 6, 14, 20, 25, 41, 49, 55, 60 and Individual Data: Appendix 2, Tables 4, 10, 17, 35, 41, 45 and 48
- Mortality
Observations were made twice daily at least 6 hours apart.
- Clinical examinations
- Time schedule: Clinical observations were conducted and recorded at least once daily, approximately 1-2 hours post dosing, throughout the course
of the study.

BODY WEIGHT: - See attached Summary Tables 2, 3, 10, 11, 16, 17, 21, 22, 37, 38, 45, 46, 51, 52, 56 and 57 and Appendix 2, Tables 2, 8, 15, 31,
32, 33, 39, 43 and 46.
- Time schedule for examinations:
Individual body weights of the male F0 and F1 rats were determined and recorded on the first day of dosing and weekly through the
pre breed exposure and mating periods.
The body weights of male F0 and F1 rats were recorded in the same manner after the mating period until scheduled euthanasia.
The body weights of female F0 and F1 rats were recorded in the same manner until evidence of mating was observed.
During gestation, sperm-positive (presumed pregnant) females were weighed on gestational days (GD) 0, 7, 14, and 20.
Dams producing litters were weighed on lactational days 0, 4, 7, 14, and 21.

FOOD CONSUMPTION AND EFFICIENCY:
- See attached Summary Tables 4, 5, 12, 13, 18, 19, 23, 24, 39, 40, 47, 48, 53, 54, 58 and 59 and Individual Data: Appendix 2,
Tables 3, 9, 16, 34, 40, 44and 47.
Individual F0 and F1 male and female feed consumption was measured weekly until mating.
Feed consumption was not recorded during the 2-week mating period.
Following mating, male feed consumption was measured on a weekly basis until the scheduled necropsy.
During pregnancy of F0 and F1 females, feed consumption was recorded on gestation days 0, 7, 14, and 20.
During lactation of F1 and F2 litters, maternal feed consumption was measured on 0, 4, 7, 14, and 21.
- Food efficiency:
Percent food efficiency (body weight gained as a percentage of food consumed) was also calculated

WATER CONSUMPTION:
Not applicable

OPHTHALMOSCOPIC EXAMINATION:
Not applicable

HAEMATOLOGY AND CLINICAL CHEMISTRY:
Not applicable
- Time schedule for collection of blood:
Not applicable
- Anaesthetic used for blood collection:
Not applicable.
- Animals fasted:
Not applicable.
- How many animals:
Not applicable.

URINALYSIS:
Not applicable

NEUROBEHAVIOURAL EXAMINATION:
Not applicable.
- Time schedule for examinations:
Not applicable.
Behavioural assessment)
Not applicable.
(Functional Performance Tests)
Not applicable.
(Sensory Reactivity)
Not applicable.
- Dose groups that were examined:
Not applicable.

OTHER:
- MATING
Animals were paired on a 1 male: 1 female basis
Vaginal smears were performed daily on each F0 and F1 female for 21 days prior to pairing and continuing from the day after
pairing until evidence of mating was observed or until the end of the mating period. The slides from the 21 days prior to mating were
evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female according to test facility SOPs.
Vaginal smears were also performed on the day of necropsy to determine the stage of estrus at demise.
- see attached Summary Tables 15, 32 and 50 and Individual Data: Appendix 2, Tables 11, 28, 42 and 59

PREGNANCY AND PARTURITION
During the period of expected parturition, the females were observed at least twice daily for initiation and completion of parturition.
On the day parturition was completed (PND 0), pups were sexed and examined for gross malformations, and the numbers of
stillborn and live pups were recorded.
Individual gestation length was calculated using the date delivery completed.
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.
Sperm parameters (parental animals):
Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0 and F1
males as appropriate.
Litter observations:
Clinical observations, body weights, and sexes for F1 and F2 pups were recorded at appropriate intervals. For both generations (F1
and F2), 10 pups per litter (5 per sex, when possible) were arbitrarily selected on PND 4 to reduce the variability among the litters.
Offspring (28/sex/group) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation.
Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats.
Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats.
Postmortem examinations (parental animals):
Each F0 and F1 parental animal received a complete detailed gross necropsy (the surviving female parental animals were
necropsied on LD 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility),
morphology, and number] were recorded for F0 and F1 males as appropriate, and ovarian primordial follicle counts and the corpora
lutea counts were recorded for all F1 females in the control and high-dose groups. Designated tissues from the F0 and F1 parental
animals were examined microscopically.
Postmortem examinations (offspring):
Non selected F1 pups were necropsied on PND 21, and F2 pups were necropsied on PND 21. Selected organs (brain, spleen, and
thymus) were weighed from 1 pup/sex/litter (when possible) from both F1 and F2 pups that were necropsied on PND 21.
Statistics:
STATISTICAL METHODS
See Attachment “"Report Text" pages 34 to 35.
Reproductive indices:
For the Mating, fertility, and gestational indices see attached ""Report Text" page 25.
Offspring viability indices:
See attached Report Text Pages 1 to 74, Results Tables and Appendices.
Clinical signs:
effects observed, treatment-related
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:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL OBSERVATIONS AND MORTALITY
PREBREED EXPOSURE PERIOD
Summary Data: Tables 1, 6, 8, 14, 33, Individual Data: Appendix 2, Tables 1, 4, 7, 10, 29.
There were no deaths attributed to test substance administration noted through the F0 prebreed exposure period (Days 0 through 70). Seven unscheduled deaths (3 found dead and 4 euthanized for humane reasons) occurred on study through Day 70. Male No. 153 (Group 4) and Female No. 152 (Group 4) were found dead on Day 1 and Day 68, respectively. The cause of death for these animals was not conclusively determined from the gross necropsy. Red staining around the nose and left eye (Male No. 153) or nose and mouth (Female No. 152), mottled (Male No. 153, correlated with alveolus hemorrhage) or discolored/dark (Female No.152) lungs, and reddish, dark fluid in the intestines (Female No. 152 only) indicate that aspiration of the test item is the most likely cause of death of these animals. Female No. 154 (Group 4) was found dead on Day 27, approximately 1.2 hours postdose, due to an apparent dosing error, as evidenced by red fluid around the nose and mouth, lungs that failed to collapse and were discolored (dark), and fluid expelled from the trachea with slight pressure applied to lungs.

Male No. 7 (Group 1), Male No. 185 (Group 4), and Female No. 124 (Group 3) were euthanized on Days 56, 66, and 70, respectively. Gross observations at necropsy (mottled/discolored lungs, red/dark fluid in stomach and gastrointestinal tract, red staining around mouth, and/or red fluid in mouth) indicate most likely aspiration of the test item, leading to the clinical signs that prompted humane euthanasia of these animals; microscopic lung findings for Male No. 7 and 185 further support this conclusion. In addition, Male No. 143 (Group 3) was euthanized for humane reasons on Day 16 due to bilateral lesions on the neck that worsened despite veterinary intervention. All other animals survived to Day 70. There were no test item-related clinical observations from Day 0 to 70. All clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Two males (Nos. 115 and 127, Group 3) exhibited neck lesions that required veterinary intervention; the neck lesions healed by Day 70.

MATING, MALE POSTMATING, AND FEMALE GESTATION AND LACTATION PERIODS
Summary Data: Tables 6, 14, 20, 25, 33 Individual Data: Appendix 2, Tables 4, 10, 14, 17, 29.
For the F0 males, there were no deaths during the mating/postmating period (Days 70 through 107). There was an increased incidence of rooting in animals given 750 mg/kg/day and of chromodacryorrhea (eye) in animals given the 200 and 750 mg/kg/day compared with the control group. All other clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Male No. 127 (Group 3; terminated as scheduled on Day 106) exhibited a neck lesion that required veterinary intervention on Days 97 to 105; this same animal had previous neck lesions requiring veterinary intervention that had healed by Day 72.

For the F0 females, there were no deaths attributed to the test substance administration during the mating, gestation, and lactation periods (Days 70 to 128); there were 3 unscheduled F0 female deaths during this time. Female Nos. 38 (Group 1) and 180 (Group 4) were severely injured by their mating partner and were therefore euthanized on Day 72 for humane reasons (both were sperm negative on Day 72 prior to euthanasia). There were no macroscopic necropsy findings for these 2 females other than the wound inflicted by the mating partner. Female No. 166 (Group 4) was euthanized for humane reasons on Day 95 (GD 22) due to dystocia with concomitant hypothermia. Macroscopic necropsy findings for Female No. 166 included pale, mottled, mildly enlarged adrenal glands and mottled lungs (all lobes), and there were 18 visible implant sites that corresponded to 18 pups (3 live and 15 dead) in the uterus.

From Day 71 through Day 113, there were no test item-related clinical observations in the F0 females that remained sperm negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. For sperm-positive F0 females, there were occasional observations of struggling during dosing in animals given 200 and 750 mg/kg/day, and there were very occasional observations of rooting post dosing and salivating prior to dosing in up to 2 females given 750 mg/kg/day during the gestation period (GD 0 through 23). Likewise, there were occasional observations of animals struggling during dosing in the groups given 200 and 750 mg/kg/day during the lactation period. One dam (No. 118) given 200 mg/kg/day had an eye injury and swollen nose from LD 17 to 21 that were not test item-related. All other clinical findings during gestation and lactation in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals or single occurrences, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

REPRODUCTIVE PERFORMANCE
Summary Data: Tables 15, 26 Individual Data: Appendix 2, Tables 6, 11, 18, 19.
No test item-related effects on F0 reproductive performance were observed at any dosage level. There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male mating indices were 69.6%, 80.0%, 95.8%, and 90.9% and female mating indices were 70.8%, 80.0%, 95.8%, and 90.9% in the control, 50, 200, and 750 mg/kg/day groups, respectively. Historical control data for male and female mating indices ranged from 87.5% to 100% and 88% to 100%, respectively. Following a thorough investigation, which included a detailed review of all the environmental data during the study and discussions with the animal vendor, the reason for the low mating indices in the control and low-dose groups remained unknown; however, 2 males (Nos. 3 and 41) were infertile based on andrology parameters, thus partially accounting for the lower mating indices in the control group. Male fertility indices were 100.0%, 90.0%, 95.7%, and 95.0% and female fertility indices were 100.0%, 90.0%, 95.7%, and 95.0% in the same respective groups. All fertility indices were within the historical control data ranges of 75.9% to 100% for F0 males and 76.7% to 100% for F0 females.

All F0 females evaluated during the final 3 weeks of the 10-week prebreed period were noted to be cycling, with a similar number and percent of females with abnormal or irregular cycles noted across all groups, including the control. There was no difference in mean cycle length (3.8 to 4.0 days) or number of cycles (4.4 to 4.6) in test item-treated groups compared with control values (3.9 days and 4.4, respectively). The mean number of days between pairing and coitus (i.e., the precoital interval) in the test item treated groups was similar to the control group values of 2.4 days.

GESTATION LENGTH AND PARTURITION
Summary Data: Table 26 Individual Data: Appendix 2, Table 19.
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Mean F0 gestation lengths in the test item-treated groups were similar to the control group value. The mean gestation lengths in the groups given 50, 200, and 750 mg/kg/day were 22.4, 22.2, and 22.4 days, respectively, compared to the mean gestation length of 22.3 days in the concurrent control group. Gestation indices were 94.4%, 95.5%, and 89.5% in the groups given 50, 200, and 750 mg/kg/day, respectively, and were not statistically significantly different from the control group value of 100.0% and were within the historical control data range of 87% to 100%. There were corresponding increases and decreases in the stillbirth index and live birth index, respectively, for groups given 200 and 750 mg/kg/day. These slight changes in the live born gestation index, stillbirth index, and live birth index in the group given 750 mg/kg/day were due to Female No. 166 being euthanized on GD 22 due to dystocia and Female No. 200 having an all-stillborn litter. Likewise, the slight changes in these same indices for the group given 200 mg/kg/day were due to Female No. 128 having a presumed all-stillborn litter, and the slight decrease in live born gestation index for the group given 50 mg/kg/day was due to Female No. 86 not delivering any pups, though having 5 visible implant sites at necropsy. No statistically significant differences were noted between the control and test item-treated groups in the number or percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically significant differences in the mean number of implantation sites, percent post implantation loss, pups delivered (live, dead, and total), and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in the group were noted.

BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 2, 3, 4, 5, 10, 11, 12, 13, Individual Data: Appendix 2, Tables 2, 3, 8, 9.
There were no adverse test item-related effects on body weights, body weight changes, or feed consumption during the prebreed exposure period (Days 0 to 70). Slight, nondose-related increases in feed consumption (g/day and g/kg/day) were noted for males
and females at several intervals (occasionally statistically significant) for all test item-treated groups when compared with the control group; however, these changes in feed consumption did not result in or correlate with statistically significant changes in body weights, body weight changes, or food efficiency compared with the controls. There were no adverse test item-related effects on F0 male body weights and body weight changes from Days 70 to 105 and feed consumption from Days 84 to 105. A statistically significant increase in feed consumption (g/day and g/kg/day) for one interval (Days 84 to 91) was noted for the F0 males given 750 mg/kg/day; however, this change in feed consumption did not result in or correlate with statistically significant effects on body weights, body weight changes, or food efficiency compared with the controls. In general, there were no notable test item-related effects on body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency from Days 84 to 112 for those F0 females that were never found sperm positive or that never delivered a live litter. Occasional statistically significant changes were observed in these parameters, but were not considered biologically significant as they were sporadic and not dose related.

GESTATION
Summary Data: Tables 16, 17, 18, 19, Individual Data: Appendix 2, Tables 12, 13.
During gestation, there were no adverse test item-related changes in F0 female body weights and feed consumption (g/day and g/kg/day). Compared with the concurrent control group, there was a statistically significant decrease (~17%) in percent food efficiency from GD 7 to 14 for the females given 750 mg/kg/day, which correlated with a decrease (~16%) in body weight change that was not statistically significant for this same group during the same interval. The following interval (GD 14 to 20) also showed a slight, though not statistically significant, decrease in percent food efficiency and body weight change (~10% and ~8%, respectively) for the females given 750 mg/kg/day compared with the concurrent control group. Thus, percent food efficiency and body weight change were slightly reduced (~8% and ~7%, respectively) between GD 0 and 20 but did not achieve statistical significance for the females given 750 mg/kg/day compared with the control group; thus, these changes were considered incidental to treatment and not adverse.

LACTATION
Summary Data: Tables 21, 22, 23, 24, Individual Data: Appendix 2, Tables 15, 16.
During lactation (LD 0 to 21), there were no notable test item-related or statistically significant changes in F0 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared
with the control group.

ANDROLOGY
Summary Data: Table 7, Individual Data: Appendix 2, Table 6.
No test item-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in males at any dosage concentration. Differences from the
control group were slight and were not statistically significant. As per the protocol, assessments using the frozen right testis, as well as sperm morphology evaluations, were conducted initially on the control and high-dose groups only. As there were no test itemrelated effects in the group given 750 mg/kg/day, evaluation of the lower dose groups was not conducted for these endpoints.

ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 7, 8, 26, 32, 33, Individual Data: Appendix 2, Tables 5, 7, 19, 28, 29.
The only test item-related macroscopic finding in F0 parental animals was noted in the lungs of the males given 200 and 750 mg/kg/day. At the F0 male necropsy, 7 males given 750 mg/kg/day were noted as having pale lungs (cranial lobe, left or bilateral), and this gross finding correlated with an increase in both absolute and relative lung weights in this dose group. Two males given 200 mg/kg/day also had pale lungs (cranial lobe, left); however, there was no significant increase in lung weights in this dose group. The thymus of 2 males (Nos. 187 and 199) given 750 mg/kg/day had multiple, pinpoint, red foci. There was an increased incidence of mottled kidneys (bilateral) in the males given 200 and 750 mg/kg/day (10 of 24 and 9 of 23 animals, respectively) compared with
the control and low-dose groups (5 of 24 and 5 of 25 animals, respectively). The testes of 2 control males (Nos. 3 and 41) were found to be reduced in size (bilateral) and flaccid (bilateral), and the epididymides of Male No. 3 were also reduced in size. Neither
of these males (Nos. 3 and 41) sired a litter and both had few motile or progressively motile sperm (thus the reason for the larger standard error in these parameters for the control group compared with the test item-treated groups). All other macroscopic gross
findings were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. There were no test item-related macroscopic findings for the F0 females necropsied on LD (≡PND) 21. All macroscopic gross findings for F0 females at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus, metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups were slight and not statistically significant.

ORGAN WEIGHTS
Summary Data: Tables 7, 32, Individual Data: Appendix 2, Tables 5, 27.
There was an increase in both absolute and relative lung weights (38% and 33%, respectively) in F0 males given 750 mg/kg/day. Absolute and relative lung weights were also statistically significantly increased (14.0% and 12.8%, respectively) in F0 females given 750 mg/kg/day compared with the control group. The increased lung weights correlated with the macroscopic (males) and microscopic (males and females) lung findings in rats given 750 mg/kg/day. Although there was a statistically significant increase (approximately 10%) in the absolute lung weight for F0 males given 50 mg/kg/day, there was no significant increase in relative lung weight and no macroscopic lung findings in this same group, as well as no significant changes in absolute and relative lung weights for F0 females given 50 mg/kg/day and F0 males and females given 250 mg/kg/day; therefore, the change in absolute lung weight for F0 males given 50 mg/kg/day was considered spurious.

MICROSCOPIC EXAMINATION
Summary Data: Tables 9, 34, Individual Data: Appendix 3.
A test item-related histopathological lesion was identified in the lungs. Chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F0 males and females given 750 mg/kg/day compared with the controls. The increased lung
weights correlated with the chronic interstitial/alveolus inflammation observed microscopically. This lesion was characterized by a spectrum of lesions which were severity dependent and included the presence of foamy to highly vacuolated macrophages within alveolar spaces, thickened alveolar septae due to the infiltration of primarily mononuclear inflammatory cells and hypertrophy/hyperplasia of alveolar epithelial cells, edema, slight congestion and/or hemorrhage, occasional infiltration of polymorphonuclear inflammatory cells, fibrosis, increased perivascular cuffing by mononuclear inflammatory cells and a variable hyperplasia of the bronchiolar associated lymphoid tissue (BALT). This inflammatory change was graded on a 5-grade severity scale where 1 was the most minimal change noted and 5 was the most severe. Most test itemexposed animals had a severity of mild to moderately severe (2-4). The overall lung severity grade was an average from both the left and right lobes. Inflammatory changes were usually focal to multifocal. Only when the severity was grade 3 to 4 did the inflammatory foci tend to merge and become confluent. The left lung tended to have more lesions and more severe lesions than the right lobe. Lesions, in general, were highly airway-centric, located near the terminal portions of the airways. This appearance was suggestive of chemical exposure through aspiration. In other areas, this determination was more difficult but may have been influenced by sectioning.

There did not appear to be any significant difference between male and female lungs with regard to the appearance of interstitial/alveolus inflammation. Another similar pulmonary finding (alveolar macrophage aggregation, alveolus) was noted in control male and female lungs and was characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes associated with it. This finding can be seen as a spontaneous change in the lungs of rats, though one cannot rule out the possibility that this finding is related to dosing with the corn oil vehicle. An equivocal, non-adverse histopathological lesion was identified in the kidneys. Renal tubule mineralization was observed in the kidneys of 7 of 23 males given 750 mg/kg/day. This lesion was characterized by the presence of multifocal mineralized renal tubules (proximal convoluted tubules) in the outer cortex. Similar appearing tubules were not seen in any of the 24 male F0 controls. The renal tubules were characterized as basophilic tubules with mineralization of both the nucleus and cytoplasm affected. Some tubules were strongly mineralized and others less.

There was no apparent cause for the mineralization and no histopathological reaction to the mineralized tubules was noted. In the study pathologist’s experience, these tubules appeared similar to artifactual changes occasionally observed as a result of tissue
fixation and/or processing. Since mineralized tubules may be an artifactual change and were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal. Other changes in the kidneys such as chronic progressive nephropathy and renal tubule regeneration were not clearly increased in either incidence or in severity and were therefore not deemed test item-related. The microscopic examination of reproductive tissues for F0 animals suspected of reduced fertility in the low- and mid-dose groups did not have any relationship to test item administration. A number of other histopathological findings were noted in a number of tissues which were typical of the spontaneous type of microscopic pathology that may be observed in this strain and age of rat.
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No adverse treatment-related effects observed at the highest dose tested.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
LITTER DATA F1
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: Table 26, Individual Data: Appendix 2, Tables 19, 20.
The mean number of pups delivered (live, dead, and total); number of live pups per litter on PND 0, 4, 7, 14, and 21(for litters
having at least 1 live pup); percentage of males per litter on PND 0, 4, 7, 14, and 21; lactational index; and postnatal survival
indices (4-, 7-, 14-, and 21-day) were unaffected by the test item at all dosage concentrations. Differences from the control group
were slight and were not statistically significant. The slight reduction (which was not statistically significant) in the 4-day survival
index per litter in the group given 750 mg/kg/day was because the entire litter for Female No. 198 was found dead (3 pups) or
euthanized (12 pups) prior to PND 2 due to poor clinical condition (including no milk band, cold to touch, reduced activity, and loss
of skin elasticity) due to the lack of care for the pups by the dam.

GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: Table 28, Individual Data: Appendix 2, Tables 20, 23.
The numbers of F1 pups found dead, euthanized, and/or missing, as well as the general physical condition of all F1 pups in this
study, were unaffected by test item administration. Pups that were found dead or euthanized (prior to scheduled euthanasia on
PND 21) numbered 4, 6, 12, and 19 in the control, 50, 200, and 750 mg/kg/day groups, respectively. One litter (Dam No. 198)
accounted for 15 of the 19 pups in the group given 750 mg/kg/day. In addition, 3, 3, 2 and 0 pups in the same respective groups
were either missing (and presumed dead) or cannibalized.
ANOGENITAL DISTANCE
Summary Data: Table 27, Individual Data: Appendix 2, Table 21.
The anogenital distances (absolute and adjusted for body weight as the covariate) in the F1 male and female pups delivered from
dams given 50, 200, and 750 mg/kg/day were similar to the respective control group values. Differences from the control group
were slight and not statistically significant.

OFFSPRING BODY WEIGHTS
Summary Data: Table 27, Individual Data: Appendix 2, Table 22.
Mean male and female pup body weights and body weight changes in the groups given 50, 200, and 750 mg/kg/day were
unaffected by test item administration throughout the postnatal period.
No statistically significant differences from the control group were noted.

NECROPSIES OF PUPS STILLBORN, FOUND DEAD, OR EUTHANIZED
Summary Data: Tables 28, 29, Individual Data: Appendix 2, Tables 23, 24.
The numbers of pups (litters) stillborn, found dead, or euthanized from PND 0 through the selection of the F1 generation on PND 21
numbered 4(4), 8(5), 12(6), and 19(5) in the control, 50, 200, and 750 mg/kg/day groups, respectively. No internal findings that
could be attributed to parental administration to the test item were noted at the necropsies of pups that were stillborn, found dead,
or euthanized.
The combined incidence of bilateral and unilateral hydronephrosis in stillborn pups was not different from the control group, nor was
the incidence of hydronephrosis seen in pups necropsied as scheduled on PND 21 different in test item-treated groups compared
with the control group; therefore, the increased pup and litter incidence of bilateral hydronephrosis in stillborn pups in both the 200
and 750 mg/kg/day dose groups compared with the control group was not considered test item-related.
There were several F1 pups found dead and euthanized moribund in the 200 mg/kg/day and/or 750 mg/kg/day groups, with
observations of no milk in the stomach, indicating the pups were not nursing. There were no F1 pups found dead in the control
group and no F1 pups euthanized moribund in the control and 200 mg/kg/day dose groups, so a direct comparison of the incidence
of this macroscopic finding across groups in unscheduled death pups is not possible. The one low-dose pup euthanized moribund
also had no milk in the stomach and loss of skin elasticity noted at necropsy. Of the 12 pups euthanized moribund in the high-dose
group (all from Dam No. 198), 3 had distended ureter (either unilateral or bilateral); however, the incidence of this finding in the
culled pups from all dose groups was similar, and thus this finding was not considered to be test item-related.

NECROPSIES OF WEANLINGS - PND 21
MACROSCOPIC EXAMINATION
Summary Data: Table 31, Individual Data: Appendix 2, Table 26.
At the PND 21 necropsy of F1 weanlings, there were no findings in the test item-treated groups that were not also observed with
similar or higher incidence in the control group.

ORGAN WEIGHTS
Summary Data: Table 30, Individual Data: Appendix 2, Table 25.
There were no statistically significant changes in F1 pup final body weights and organ weights compared with the control group.
Slight decreases in absolute and relative spleen weights (approximately 9% to 10% and 4% to 5%, respectively) were observed in
F1 pups in the group given 750 mg/kg/day compared with the control group. The spleen weights in F1pups from the high-dose
group in this study fall within the historical control data ranges for this parameter and were therefore considered an equivocal, non-adverse finding.

MICROSCOPIC EXAMINATION
Summary Data: Table 31, Individual Data: Appendix 3.
There were no microscopic findings in the F1 PND 21 weanlings in the test item-treated groups that were not also observed with
similar or higher incidence in the control group.

F1 DEVELOPMENTAL LANDMARKS
BALANOPREPUTIAL SEPARATION
Summary Data: Table 36, Individual Data: Appendix 2, Table 31.
The test item-related increase in attainment of balanopreputial separation in males given 750 mg/kg/day was not considered
adverse, as the day of attainment falls within the historical control ranges for this parameter. The mean ages of attainment of
balanopreputial separation were 43.3, 43.2, and 43.2 days in the groups given 50, 200, and 750 mg/kg/day, respectively, compared
with 41.6 in the control group, and were all statistically significantly longer than the control group, though in a nondose-responsive manner and within the historical control range for the mean age of attainment of balanopreputial separation of 40.8 to 43.6 days.
Mean body weights at the age of attainment were 254.0, 249.8, and 245.4 g in the same respective groups, compared with 238.9 g
in the control group, and were not statistically significant. When adjusted with body weight as a covariate, the adjusted mean ages
of attainment of balanopreputial separation were 43.0, 43.1, and 43.3 days in the groups given 50, 200, and 750 mg/kg/day,
respectively, compared with 42.0 in the control group, and although dose-responsive, only the group given 750 mg/kg/day was
statistically significantly longer than the control group. This test item-related increase in adjusted age of attainment of
balanopreputial separation was not considered adverse, as 43.3 days falls within the historical control ranges for this parameter of
40.7 to 43.8 days. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group
with the longest delay in attainment of balanopreputial separation were similar to the group mean data, further supporting the
conclusion that the delay in preputial separation was non-adverse.

VAGINAL PATENCY
Summary Data: Table 36, Individual Data: Appendix 2, Table 32.
Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test item
administration. The mean ages of attainment of vaginal patency were 31.3, 30.6, and 30.0 days in the groups given 50, 200, and
750 mg/kg/day, respectively compared with 29.9 days in the control group. Mean body weights at the age of attainment were 117.5,
111.0, and 105.5 g in the same respective groups compared with 108.6 g in the control group. The mean age for the group given 50
mg/kg/day was statistically significant, though was not considered test item-related as there was no dose-response and the
adjusted mean age was not statistically significantly different from the control group.

GENERATION F1
CLINICAL OBSERVATIONS AND MORTALITY
PREBREED EXPOSURE PERIOD
Summary Data: Tables 35, 41, 43, 49, 68, Individual Data: Appendix 2, Tables 30, 35, 38, 41, 60.
There were no deaths directly attributed to the test substance administration noted through the F1 weaning and prebreed exposure
period (Days -20 through 70). Seven unscheduled deaths (6 found dead and 1 euthanized for humane reasons) occurred through
Day 70.
Male No. 1147 (Group 2, 50 mg/kg/day) was found dead on Day -2 (PND 36) after being noted as lethargic the previous day. Male
No. 1305 (Group 4, 750 mg/kg/day) was also found dead on Day -2 (PND 38). The cause of death for these 2 males could not be
determined upon necropsy; there were no obvious signs of gavage error and some autolysis was present. Group 4 Male Nos. 1329
and 1351 were found dead on Days 2 (PND 41) and 3 (PND 40), respectively. Both males had hydronephrosis of the right kidney
and dark red lungs. The lungs from Male No. 1351 failed to collapse and had pale areas around the edges of all lobes. Slight
pressure on the lungs released bubbles from the trachea, suggesting the cause of death of Male No. 1351 was a gavage error.
Based on the appearance of the lungs at necropsy, Male No. 1329 may have died due to aspiration of the dose formulation. Group
4 Female Nos. 1346 and 1354 were found dead on Days 18 (PND 56) and 30 (PND 67), respectively. Female No. 1346 had
struggled during dosing the day prior to being found dead. At necropsy, the only finding was red substance in the nose and mouth.
The cause of death of Female No. 1346 could not be determined upon necropsy. Female No. 1354 was noted as lethargic at the
postdose observation time and died a short time later. At necropsy, slight hydronephrosis of the right kidney was seen, and the
lungs were found to be red and failed to collapse fully. Slight pressure on the lungs released fluid and bubbles from the trachea,
suggesting the cause of death of Female No. 1354 was gavage error.
Female No. 1348 (Group 4, 750 mg/kg/day) was euthanized for humane reasons on Day 54 (PND 91) on welfare grounds. At
necropsy, reddish fluid from nose and mouth, reddish fluid in the intestines, very little food present in the stomach, and dark red
lungs that did not fully collapse were noted, suggesting the apparent cause of death was gavage error. All other animals survived to
Day 70.
There was a slightly increased incidence of rooting in male and female F1 animals given 750 mg/kg/day and of chromodacryorrhea
(eye) in females given 200 mg/kg/day and 750 mg/kg/day compared with the control group. These observations are not adverse. All
other clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single
animals, were limited to unscheduled death animals, were not noted in a dose-related manner, and/or were common findings for
laboratory rats of this age and strain.

MATING, MALE POSTMATING, AND FEMALE GESTATION AND
LACTATION PERIODS
Summary Data: Tables 41, 43, 49, 55, 60, 68, Individual Data: Appendix 2, Tables 35, 38, 41, 45, 48, 60.
For the F1 males, there was one death during the mating/postmating period (Days 70 through 107). Male No. 1325 (Group 4, 750
mg/kg/day) was noted as lethargic and gasping for breath prior to dying on Day 78. At necropsy, the lungs were mottled and failed
to fully collapse; when slight pressure was applied to the lungs, bubbles were released from the trachea. The apparent cause of
death of this animal was either a direct dosing error or inadvertent aspiration of the test item leading to a chronic aspiration
pneumonia.
All clinical findings in the F1 male, test item-treated groups were noted with similar incidence in the control group, were limited to
single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
For the F1 females, there were no deaths directly attributed to the test substance administration during the mating, gestation, and
lactation periods. Female No. 1352 (Group 4, 750 mg/kg/day) was found dead on Day 96 (LD 1) with the macroscopic necropsy
finding of dark red substance present in the uterus. On the previous day (LD 0), clinical signs for Animal No. 1352 included
piloerection, dystocia, labored breathing, lethargy, and excess red substance present; as the animal was not hypothermic and was
actively delivering pups at the time of evaluation by the veterinarian, the veterinarian recommendation was for continued
monitoring.
From Day 71 through Day 113, there were no test item-related clinical observations in the F1 females that remained sperm
negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to
single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. For sperm-positive F1 females, there were no test item-related clinical observations during the gestation period (GD 0 through 23)
or lactation period (LD 0 through 21). One dam (No. 1316) given 750 mg/kg/day a mass/sore on the chest for the entire lactation
period and from days 9 (mass) or 18 (sore) through 21 of gestation. The area was not seen at terminal necropsy and thus was not
saved for evaluation. All other clinical findings during gestation and lactation in the test item-treated groups were noted with similar
incidence in the control group, were limited to single animals or single occurrences, were not noted in a dose-related manner,
and/or were common findings for laboratory rats of this age and strain.

REPRODUCTIVE PERFORMANCE
Summary Data: Tables 50, 61
Individual Data: Appendix 2, Tables 42, 49, 50
No test item-related effects on F1 reproductive performance were observed at any dosage level.
There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male
mating indices were 92.9%, 92.6%, 85.7%, and 88.0% and female mating indices were 92.9%, 92.9%, 85.7%, and 88.0% in the
control, 50, 200, and 750 mg/kg/day groups, respectively. Historical control data male and female F1 mating indices ranged from
70.0% to 100% and 50% to 100%, respectively. Male fertility indices were 96.2%, 92.0%, 95.8% and 81.8% and female fertility
indices were 96.2%, 92.3%, 95.8% and 81.8% in the same respective groups. Although the fertility index for males and females
was lower in the group given 750 mg/kg/day, all fertility indicies were within the historical control data ranges of 76.2% to 100% for
F1 males and 70.8% to 100% for F1 females.
All test item-treated F1 females evaluated during the final 3 weeks of the 10-week pre breed period were noted to be cycling
compared with 89.3% of the control females noted to be cycling.
There was no statistically significant difference in the number and percent of test item-treated females with abnormal or irregular
cycles compared with the control group. There were no differences in mean cycle length (4.1 to 4.3 days) and number of cycles (4.0
to 4.4) in test item treated groups compared with control values (4.0 days and 4.6, respectively). The mean numbers of days
between pairing and coitus (i.e., the precoital interval) in the test item-treated groups were similar to the control group values of 2.8
days.

GESTATION LENGTH AND PARTURITION
Summary Data: Table 61, Individual Data: Appendix 2, Table 50.
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration.
Gestation indices were 100%, 100%, and 88.9% in the groups given 50, 200, and 750 mg/kg/day, respectively, and were not
statistically significantly different from the control group value of 96.0%. The historical control data range for F1 gestational index is
94.1% to 100%. The slight, though not statistically significant, decrease in the liveborn gestation index in the group given 750
mg/kg/day was due to Female Nos. 1324 and 1344 having implantation sites but not delivering a litter. There was also one control
female (No. 1024) that had implantation sites but did not deliver a litter. Therefore, these observations are not considered test item
related. No statistically significant differences were noted between the control and test item-treated groups in the number or
percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically
significant differences in the mean number of implantation sites, percent postimplantation loss, pups delivered (live, dead, and
total), stillbirth index, live birth index, and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in
the group were noted.

BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 37, 38, 39, 40, 45, 46, 47, 48, Individual Data: Appendix 2, Tables 33, 34, 39, 40.
There were no adverse, test item-related effects on F1 body weights or feed consumption during the prebreed exposure period
(Days 0 to 70). The increased (7% to 11%) body weights for F1 females given 50 mg/kg/day, though statistically significant at every
interval from Day 0 to Day 70, was not considered test item-related since there was no dose relationship and only occasional
corresponding significant increases in body weight change, food consumption (g/day only), and food efficiency. Although other
occasional statistically significant changes in body weight, body weight change, food consumption, and food efficiency were noted
when compared with the control group, including the slightly decreased (4.4% and 5.4%) percent food efficiency for the overall
interval from Days 0 to 70 for F1 males given 50 and 750 mg/kg/day, respectively, these changes were not considered the result of
test item administration since these changes were sporadic, slight, not dose related, and generally had no effect on the other body
weight and/or food consumption parameters.
There were no adverse test item-related effects on F1 male body weights and body weight changes from Days 70 to 105 and feed
consumption from Days 84 to 105. Statistically significant decreases in body weight change and percent food efficiency for one
interval (Days 91 to 98) were noted for the F1 males given 750 mg/kg/day; however, these changes did not result in or correlate
with statistically significant effects on body weight or feed consumption (g/day and g/kg/day) compared with the controls for this
same interval and were thus considered nonadverse. The statistically significant increases in body weight change and/or percent
food efficiency during the postmating holding period, for F1 males given the lower dose levels, were sporadic and not dose related,
and therefore not considered test item related.
There were no statistically significant changes in body weights, body weight changes, feed consumption (g/day and g/kg/day), and
percent food efficiency from Days 84 to 112 for those F1 females that were never found sperm positive or that never delivered a live
litter.

GESTATION
Summary Data: Tables 51, 52, 53, 54, Individual Data: Appendix 2, Tables 43, 44.
There were no test item-related changes in body weights, body weight changes, feed consumption (g/day and g/kg/day), and
percent food efficiency in the test item-treated groups compared with the control group during gestation. Although body weights for
females given 50 mg/kg/day were statistically significantly increased at each interval, there were no corresponding statistically significant increased in body weight change, feed consumption, or percent food efficiency. The change in body weights was not
considered test item-related as there was no dose response.

LACTATION
Summary Data: Tables 56, 57, 58, 59, Individual Data: Appendix 2, Tables 46, 47.
During lactation (LD 0 to 21), there were no notable test item-related changes in F1 female body weights, body weight changes,
feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group.
The one statistically significant change (an increase in body weight for females given 50 mg/kg/day on LD 21) was not considered
test item-related as the change was sporadic, showed no dose response, and did not correlate with any changes in any other body
weight or food parameters.

ANDROLOGY
Summary Data: Table 42, Individual Data: Appendix 2, Table 37
There was an equivocal, non-adverse, statistically significant increase in the percent of abnormal sperm in the F1 males given 750
mg/kg/day compared with the control group; however, the historical control data range of 1.29% to 6.1% encompasses the 2.29%
seen in the high dose in this study. There was an increase in the mean number of sperm with blunt heads (indicating the absence
of the acrosome), sperm with small heads, and sperm with heads only or tails only. One particular male in the high dose group
(Animal No. 1339) had a very high percentage of sperm with heads only; this breakage was possibly test item-related and Female
No. 1336, mated with this male, failed to become pregnant.
No test item-related effects were observed on the F1 spermatogenesis endpoints of mean testicular and epididymal sperm numbers
and sperm production rate, motility, and progressive motility in males at any dosage concentration. Differences from the control
group were slight
and were not statistically significant for these parameters.

ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 42, 43, 61, 67, 68, Individual Data: Appendix 2, Tables 36, 38, 50, 58, 59, 60.
The only test item-related macroscopic findings in F1 parental animals were observed in the lungs of males and females given 750
mg/kg/day. At the F1 male scheduled necropsy, a total of 11 males given 750 mg/kg/day were noted as having lung findings,
including small dark red spots and mottled (alone or with other findings such as firm, tan, and/or small dark spots), and these gross
findings correlated with an increase in both absolute and relative lung weights in this dose group. There was an increased incidence
of mottled kidneys (bilateral) in the males given 750 mg/kg/day (10 of 24) compared with the control group (4 of 28). All other
macroscopic gross findings in F1 males were noted with similar incidence among the groups, were limited to single animals, were
not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
At the scheduled necropsies on LD (≡PND) 21, a total of 4 F1 females given 750 mg/kg/day were noted as having lung findings
including mottled, firm, tan, for 2 animals and left cranial lobe, tan, meaty for 2 animals; these gross findings correlated with an
increase in absolute lung
weights in this dose group. There were 2 F1 females given 750 mg/kg/day that had a fluid-filled cyst in the uterus at necropsy; the
relationship to test item administration of this finding is unknown. All other macroscopic gross findings for F0 females at scheduled
necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related
manner, and/or were common findings for laboratory rats of this age and strain.
No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus,
metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups
were slight and not statistically significant.

ORGAN WEIGHTS
Summary Data: Tables 42, 67, Individual Data: Appendix 2, Tables 36, 58.
There was an increase in both absolute and relative lung weights (39.5% and 37.7%, respectively) in F1 males given 750
mg/kg/day. Absolute and relative lung weights were also increased [12.9% (statistically significant) and 6.9% (not statistically
significant), respectively] in F1 females given 750 mg/kg/day compared with the control group. The increased lung weights
correlated with the macroscopic and microscopic lung findings in rats given 750 mg/kg/day.

MICROSCOPIC EXAMINATION
Summary Data: Tables 44, 67, 69, Individual Data: Appendix 3.
Like the F0 males and females, chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F1
males and females given 750 mg/kg/day compared with the controls. All 24 F1 males and 24 F1 females given 750 mg/kg/day that
survived until scheduled euthanasia had this test item-related finding. The increased lung weights correlated with the chronic
interstitial/alveolus inflammation observed microscopically.
Also like the F0 animals, alveolar macrophage aggregation, alveolus was noted in F1 control male and female lungs and was
characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes
associated with it. This finding can be seen as a spontaneous change in the lungs of rats, though one cannot rule out the possibility
that this finding is related to dosing with the corn oil vehicle.
Test item-related slight increases in incidence of renal tubule degeneration/necrosis and renal tubule hyaline droplets were seen in
the F1 males given 750 mg/kg/day. Degeneration/necrosis was characterized by the presence of multifocal, cortical proximal
convoluted tubules which were lined by epithelial cells that were slightly vacuolated with nuclear pyknosis and containing
occasional sloughed necrotic cells. This change was very subtle and recorded as minimal. In a few instances, lightly staining
eosinophilic droplets were increased in proximal convoluted tubules of the same animals. This morphologic appearance suggested
hydrocarbon-induced α2μ-globulin male rat nephropathy. Hyaline droplets were not observed in the treated females. Kidneys from selected males given the vehicle or 750 mg/kg/day from both the F0 and F1 generations (10 males/group/generation)
were stained with Mallory Heidenhain stain which is used to characterize α2μ-globulin in renal tubules. As expected due to the age
of these rats, renal tubules from all of these selected animals contained variable numbers of tubule droplets which stained positive
and had the characteristic distribution and features of α2μ-globulin (male rats of this age contain α2μ-globulin). The droplets were
subjectively scored on a scale of 1 to 5 +. No apparent increase in positive tubule droplets was present in the F0 males given 750
mg/kg/day compared with the F0 control males. However, the mean average score of the positive tubule droplets was increased in
the F1 males given 750 mg/kg/day compared with the F1 control males, confirming that these kidney findings were the result of α2μ
-globulinergic nephropathy, which is known to occur in male rats following exposure to hydrocarbons.
Other changes in the kidneys, such as chronic progressive nephropathy and renal tubule regeneration, were not clearly increased
in either incidence or in severity and were therefore not deemed test item-related.
For F1 males and females in the low- and mid-dose groups suspected of reduced fertility, there were no test item-related
microscopic findings in the reproductive organs/tissues examined. Two males given 50 mg/kg/day and 3 males given 200
mg/kg/day had inflammation of the prostate gland; however, 7 males given the vehicle control and 9 males given the high dose of
750 mg/kg/day had a similar finding; thus, this finding was not considered test item-related. One male given 200 mg/kg/day showed
seminiferous tubule atrophy in the testis. One female given 50 mg/kg/day had dilatation of the uterus and one other had an ovarian
cyst while one female given 200 mg/kg/day had ovarian atrophy. These microscopic findings were not observed in F1 males and
females given the high dose of 750 mg/kg/day and may have contributed to the reduced fertility for these particular animals.
A number of other histopathological findings were noted in a number of tissues which were typical of the spontaneous type of
microscopic pathology that may be observed in this strain and age of rat.
Ovarian follicle counts were not statistically significantly different in the F1 females given 750 mg/kg/day compared with the control
group; therefore, counts were not conducted on the lower dose groups.

F2 LITTER DATA
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: Table 61, Individual Data: Appendix 2, Tables 50, 51.
The mean number of pups delivered (live, dead, and total); number of live pups per litter on PND 0, 4, 7, 14, and 21 (for litters
having at least 1 live pup); percentage of males per litter on PND 0, 4, 7, 14, and 21; lactational index; and postnatal survival
indices (4-, 7-, 14-, and 21-day) were unaffected by the test item at all dosage concentrations. Differences from the control group
were slight and were not statistically significant.

GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: Table 63, Individual Data: Appendix 2, Tables 51, 54.
The numbers of F2 pups found dead, euthanized, and/or missing, as well as the general physical condition of all F2 pups in this
study, were unaffected by test item administration. Pups that were found dead or euthanized (prior to scheduled euthanasia on
PND 21) numbered 17, 17, 25, and 17 in the control, 50, 200, and 750 mg/kg/day groups, respectively. One litter (Dam No. 1352
found dead on LD 1) accounted for 10 of the 17 pups in the group given 750 mg/kg/day. In addition, 3, 3, 3 and 0 pups in the same
respective groups were missing (and presumed dead).

ANOGENITAL DISTANCE
Summary Data: Table 62, Individual Data: Appendix 2, Table 52.
The anogenital distances (absolute and adjusted for body weight as the covariate) in the F2 male and female pups delivered from
dams given 50, 200, and 750 mg/kg/day were similar to the respective control group values. Differences from the control group
were slight and not statistically significant.

OFFSPRING BODY WEIGHTS
Summary Data: Table 62, Individual Data: Appendix 2, Table 53.
Mean male and female pup body weights and body weight changes in the 50, 200, and 750 mg/kg/day groups were unaffected by
parental test item administration throughout the postnatal period. No statistically significant differences from the control group were
noted.

NECROPSIES OF PUPS STILLBORN, FOUND DEAD, OR EUTHANIZED
Summary Data: Tables 63, 64, Individual Data: Appendix 2, Tables 54, 55.
The number of F2 pups (litters) stillborn, found dead, or euthanized from PND 0 through PND 21 were 17(11), 16(13), 25(13), and
17(4) in the control, 50, 200, and 750 mg/kg/day groups, respectively. No internal findings that could be attributed to parental
administration to the test item were noted at the necropsies of F2 pups that were stillborn, found dead, or euthanized.
Findings in the stillborn, found dead, or euthanized pups from test item-treated dams were similar to the findings in stillborn, found
dead, or euthanized pups from vehicle-treated dams, culled PND 4 pups, and/or scheduled death PND 21 F2 pups.

NECROPSIES OF WEANLINGS - PND 21
MACROSCOPIC EXAMINATION
Summary Data: Table 66, Individual Data: Appendix 2, Table 57.
At the PND 21 necropsy of F2 weanlings, there were no test item-related findings.
Hydronephrosis of the right kidney was noted at a similar incidence in males in the control and test item-treated groups.

ORGAN WEIGHTS
Summary Data: Table 65, Individual Data: Appendix 2, Table 56.
There were no statistically significant changes in F2 pup final body weights and organ weights compared with the control group.
Slight decreases in absolute and relative spleen weights (approximately 7% to 8% and 4% to 7%, respectively) were observed in
the F2 male and female pups in the group given 750 mg/kg/day compared with the control group. Given the small magnitude of the
change, the lack of statistical significance, and since the weights fall within the historical control data ranges, these changes were
considered equivocal and not adverse.

MICROSCOPIC EXAMINATION
Summary Data: Table 66, Individual Data: Appendix 3.
Dilatation of the pelvis of the kidney was noted for all male pups with a macroscopic finding of hydronephrosis; thus, this finding
was noted at a similar incidence in the control and test item treated groups and was therefore not considered test item-related.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental effects in offspring
Critical effects observed:
not specified
Reproductive effects observed:
not specified

OBJECTIVE AND METHODS

The objective of this study was to characterize the effects of the test substance on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrus cycle, mating behavior, conception, gestation, parturition, lactation, and weaning of the F1generation, as well as the neonatal survival, growth, and development of the F1and F2offspring. In addition, the systemic toxicity of the test substance was partially characterized in this study by evaluation of several nonreproductive tissues. Three groups of male and female Crl:CD(SD) rats (25 and 28/sex/group for the F0and F1generations, respectively) were administered the test item (CAS No. 848301-67-7), daily by oral gavage for at least 70 consecutive days prior to mating. Dosage levels were 50, 200, and 750 mg/kg/day for the F0and F1generations. A concurrent control group of 25 and 28/sex for the F0and F1generations, respectively, received the vehicle, corn oil (CAS No. 8001-30-7) on a comparable regimen. F0animals were approximately 6 to 7 weeks (42 to 46 days) of age at the initiation of test item administration. The test item was administered to offspring selected to become the F1parental generation following weaning, beginning on postnatal day (PND) 22. The F0and F1males continued to receive the test item throughout mating and through the day prior to euthanasia. The F0and F1females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia. Animals were observed at least twice daily for mortality. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for males throughout the study and for females prior to mating and during gestation and lactation. Vaginal smears were performed daily for determination of estrous cycles beginning 21 days prior to pairing. All F0and F1females were allowed to deliver and rear their pups until weaning on lactation day (LD≡PND) 21. Clinical observations, body weights, and sexes for F1and F2pups were recorded at appropriate intervals. For both generations (F1and F2), 10 pups per litter (5 per sex, when possible) were arbitrarily selected on PND 4 to reduce the variability among the litters. Offspring (28/sex/group) from the pairing of the F0animals were selected on PND 21 to constitute the F1generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1rats. Nonselected F1pups were necropsied on PND 21, and F2pups were necropsied on PND 21. Selected organs (brain, spleen, and thymus) were weighed from 1 pup/sex/litter (when possible) from both F1and F2pups that were necropsied on PND 21. Each F0and F1parental animal received a complete detailed gross necropsy (the surviving female parental animals were necropsied on LD 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0and F1males as appropriate, and ovarian primordial follicle counts and the corpora lutea counts were recorded for all F1females in the control and high-dose groups. Designated tissues from all F0and F1parental animals were examined microscopically.

Conclusions:
SUMMARY OF RESULTS
Administration of the test substance to male and female rats at dosages up to 750 mg/kg/day had no effect on reproductivevperformance or gestation length and parturition of both the F0 and F1 parental generations. In addition, there were no test itemrelated effects on F1 and F2 litter parameters, postnatal survival, physical condition/mortality, anogenital distance, and pup body weights. Vaginal patency of F1 females was unaffected by test item administration. There was a statistically significant, test articlerelated increase in the mean age and adjusted age of attainment of balanopreputial separation in F1 males given 750 mg/kg/day. However, this change was not considered adverse, as the age and adjusted age of attainment fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day,
although slightly lower than the control group, were not statistically significantly different from the control group and also fell within the historical control data range. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group, with the longest delay in attainment of balanopreputial separation, were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse. Sperm morphology assessments of F1 males showed a statistically significant increase in the percent of abnormal sperm in those males given 750 mg/kg/day; however, this change was not observed in F0 males and resulted primarily from a single male, and was therefore considered equivocal and non adverse since the percent of abnormal sperm seen fell within the historical control data range for this parameter, reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day were unaffected, and there were no microscopic findings in the testes.

Overall, there were 19 unscheduled deaths of parental animals (4 F0 males, 6 F0 females, 5 F1 males, and 4 F1 females). None of the deaths were directly attributed to the test item. Based on macroscopic (and in some cases microscopic) findings, 11 of the deaths were likely due to gavage error and/or aspiration of the dose formulation into the lungs. With the low viscosity nature of the test item and the use of corn oil as the vehicle, aspiration events were not unexpected. Two F0 females were injured by their mating partner and euthanized for humane reasons, and 1 F0 male was euthanized for humane reasons due to lesions on the neck, likely
caused by scratching at the ear tag. One F0 female given 750 mg/kg/day was euthanized due to dystocia, and 1 F1 female given 750 mg/kg/day was found dead the day after dystocia was observed. The cause of death for 3 animals (1 F1 male given 50 mg/kg/day, 1 F1 male given 750 mg/kg/day, and 1 F1 female given 750 mg/kg/day) could not be definitively determined at necropsy.

There were no adverse test item-related effects on F0 and F1 parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F0 and F1 generations and the kidneys (males only) of the F1 generation. There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F0 and F1 males and females given 750 mg/kg/day; this microscopic finding correlated with macroscopic observations in F0 and F1 males and F1 females, as well as increased absolute and relative lung weights in F0 and F1 males and females. As these lung lesions were considered to be secondary to aspiration of the dose formulation, and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day, repeat-dose study with a similar
test item, the lungs from the lowand mid-dose animals were not evaluated. In the F1 males, test item-related slight increases in renal tubule degeneration/necrosis and renal tubule hyaline droplets, suggestive of hydrocarboninduced α2μ-globulin male rat nephropathy, were seen in the males given 750 mg/kg/day. Special staining of kidneys from males in the control and high-dose group confirmed this lesion to be hydrocarbon-induced α2μ-
globulin male rat nephropathy, an anticipated outcome of this study; therefore, the kidneys of the low- and mid-dose animals were not evaluated. In the F0 males, renal tubule mineralization was noted. Since this finding may be an artifactual change resulting from tissue fixation and/or processing, and since mineralized tubules were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal and non-adverse. There were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition, mortality, pup body weights and anogenital distance. Equivocal, non-adverse decreases (not statistically significant) in absolute and relative spleen weights were observed in the F1 and F2 male and female PND 21 pups in the group given 750 mg/kg/day, compared with the control group. Macroscopic and microscopic findings in PND 21 pups were not test item related.

CONCLUSIONS
The test substance [0 (vehicle), 50, 200, or 750 mg/kg/day)] was given orally to F0 and F1 parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, post mating holding (for males), and gestation and lactation (for females) periods. For F1 males given 750 mg/kg/day, there was a test item-related, non-adverse effect on preputial separation (slight delay) and an equivocal, non-adverse effect on sperm morphology (slight increase in percent abnormal sperm); the slight changes in these parameters fell within the historical control data range. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and increased lung weights and the kidneys (renal tubule degeneration/necrosis and renal tubule hyaline droplets
confirmed to be α2μ-globulin) of F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. Additional equivocal, non-adverse findings included renal tubule mineralization in the kidneys of F0 males given 750 mg/kg/day and slightly decreased spleen weights in F1 and F2 pups. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, and the absence of adverse findings directly attributable to the test item in non reproductive tissues, a dosage level of 750 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.
Executive summary:

SUMMARY OF RESULTS AND CONCLUSION

Administration of the test substance to male and female rats at dosages up to 750 mg/kg/day had no effect on reproductive performance or gestation length and parturition of both the F0 and F1 parental generations. In addition, there were no test itemrelated effects on F1 and F2 litter parameters, postnatal survival, physical condition/mortality, anogenital distance, and pup body weights. Vaginal patency of F1 females was unaffected by test item administration. There was a statistically significant, test articlerelated increase in the mean age and adjusted age of attainment of balanopreputial separation in F1 males given 750 mg/kg/day. However, this change was not considered adverse, as the age and adjusted age of attainment fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day, although slightly lower than the control group, were not statistically significantly different from the control group and also fell within the historical control data range. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group, with the longest delay in attainment of balanopreputial separation, were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse. Sperm morphology assessments of F1 males showed a statistically significant increase in the percent of abnormal sperm in those males given 750 mg/kg/day; however, this change was not observed in F0 males and resulted primarily from a single male, and was therefore considered equivocal and non adverse since the percent of abnormal sperm seen fell within the historical control data range for this parameter, reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day were unaffected, and there were no microscopic findings in the testes. Overall, there were 19 unscheduled deaths of parental animals (4 F0 males, 6 F0 females, 5 F1 males, and 4 F1 females). None of the deaths were directly attributed to the test item. Based on macroscopic (and in some cases microscopic) findings, 11 of the deaths were likely due to gavage error and/or aspiration of the dose formulation into the lungs. With the low viscosity nature of the test item and the use of corn oil as the vehicle, aspiration events were not unexpected. Two F0 females were injured by their mating partner and euthanized for humane reasons, and 1 F0 male was euthanized for humane reasons due to lesions on the neck, likely caused by scratching at the ear tag. One F0 female given 750 mg/kg/day was euthanized due to dystocia, and 1 F1 female given 750 mg/kg/day was found dead the day after dystocia was observed. The cause of death for 3 animals (1 F1 male given 50 mg/kg/day, 1 F1 male given 750 mg/kg/day, and 1 F1 female given 750 mg/kg/day) could not be definitively determined at necropsy. There were no adverse test item-related effects on F0 and F1 parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F0 and F1 generations and the kidneys (males only) of the F1 generation. There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F0 and F1 males and females given 750 mg/kg/day; this microscopic finding correlated with macroscopic observations in F0 and F1 males and F1 females, as well as increased absolute and relative lung weights in F0 and F1 males and females. As these lung lesions were considered to be secondary to aspiration of the dose formulation, and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day, repeat-dose study with a similar test item, the lungs from the lowand mid-dose animals were not evaluated. In the F1 males, test item-related slight increases in renal tubule degeneration/necrosis and renal tubule hyaline droplets, suggestive of hydrocarboninduced α2μ-globulin male rat nephropathy, were seen in the males given 750 mg/kg/day. Special staining of kidneys from males in the control and high-dose group confirmed this lesion to be hydrocarbon-induced α2μ-globulin male rat nephropathy, an anticipated outcome of this study; therefore, the kidneys of the low- and mid-dose animals were not evaluated. In the F0 males, renal tubule mineralization was noted. Since this finding may be an artifactual change resulting from tissue fixation and/or processing, and since mineralized tubules were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal and non-adverse. There were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition, mortality, pup body weights and anogenital distance. Equivocal, non-adverse decreases (not statistically significant) in absolute and relative spleen weights were observed in the F1 and F2 male and female PND 21 pups in the group given 750 mg/kg/day, compared with the control group. Macroscopic and microscopic findings in PND 21 pups were not test item related.

CONCLUSIONS The test substance [0 (vehicle), 50, 200, or 750 mg/kg/day)] was given orally to F0 and F1 parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, post mating holding (for males), and gestation and lactation (for females) periods. For F1 males given 750 mg/kg/day, there was a test item-related, non-adverse effect on preputial separation (slight delay) and an equivocal, non-adverse effect on sperm morphology (slight increase in percent abnormal sperm); the slight changes in these parameters fell within the historical control data range. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and increased lung weights and the kidneys (renal tubule degeneration/necrosis and renal tubule hyaline droplets confirmed to be α2μ-globulin) of F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. Additional equivocal, non-adverse findings included renal tubule mineralization in the kidneys of F0 males given 750 mg/kg/day and slightly decreased spleen weights in F1 and F2 pups. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, and the absence of adverse findings directly attributable to the test item in non reproductive tissues, a dosage level of 750 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.

Endpoint:
fertility, other
Remarks:
Sub-chronic toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report equivalent or similar to OECD guideline 413
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
other: OECD TG 413
Principles of method if other than guideline:
Male/female rats were exposed to decalin vapor (0, 25, 50, 100, 200 or 400 ppm) for 6 hours plus 12 minutes/day, 5 days/week for 14 weeks. Reproductive tissue evaluations for male rats included epididymis, cauda epididymis and testis weights, spermatid measurements and epididymal sperm motility measurements. Female rats were assessed for all 4 stages of the estrous cycle.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic laboratory animals and services, Germantown, NY
- Diet (e.g. ad libitum except during exposure and urine collection periods): NTP-2000, Zeigler Bros., Inc, Gardners, PA
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 12 days (males), 13 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 26
- Relative humidity: 55 +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Vapor was generated by pumping substance through a heated glass column filled with glass beads to increase surface area for evaporation. Heated nitrogen, pumped into the column, was used to vaporize the substance before transporting to exposure chamber at elevated temperature to avoid condensation. In exposure room, vapor was mixed with additional heated air in distribution manifold (at constant pressure) before being pumped through individual temeperature-controlled delivery lines to each exposure chamber. Vapor was diluted with conditioned chamber air to achieve desired exposure concentration.

Exposure chambers were designed to maintain a uniform vapor concentration throughout the chamber (total active mixing volume of 1.7 m3/chamber). Particle detector was used in exposure chambers to monitor particle count. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) ensuring that vapor and not aerosol was produced.

Build-up and decay rates for chamber vapor concentration were determined with animals present in chambers. At a chamber airflow rate of 15 air changes/ hour, time required to achieve 90% of target concentration (T90) was determined to be 12 minutes.
Details on mating procedure:
N/A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Substance concentration in exposure chambers were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 24 minutes using a 12-port stream select valve. The on-line gas chromatograph was checked throughout the day for instrument drift against an on-line standard of decalin in nitrogen supplied by a diffusion tube standard generator. The on-line gas chromatograph was calibrated monthly by a comparison of chamber concentration data to data from grab samples, which were collected with charcoal sampling tubes, extracted with toluene containing 1-phenylhexane as an internal standard, and analyzed by an off-line gas chromatograph. The volumes of gas were sampled at a constant flow rate ensured by a calibrated critical orifice. The off-line gas chromatograph was calibrated with gravimetrically prepared standards of decalin containing 1-phenylhexane as an internal standard in toluene.
Duration of treatment / exposure:
6 hours + T90 (12 minutes)/day
Frequency of treatment:
5 days/week for 14 weeks
Details on study schedule:
Groups of 10 male and 10 female rats were distributed randomly into groups of approximately equal initial mean body weights (1 animal/cage). Additional groups of 10 male and 10 female rats were exposed to the same concentrations for 6 weeks for clinical pathology analyses; additional groups of five male rats were exposed to the same concentrations for 2 weeks for renal toxicity analyses.
Remarks:
Doses / Concentrations:
0 (control), 25, 50, 100, 200 or 400 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals/sex/group
Control animals:
yes, sham-exposed
Parental animals: Observations and examinations:
Observed twice daily; core study animals were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly.
Oestrous cyclicity (parental animals):
Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from core study females exposed to 0, 100, 200, or 400 ppm for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Sperm parameters (parental animals):
The following parameters were evaluated: spermatid heads per testis, per gram testis, per cauda, and per gram cauda and epididymal spermatozoal motility. The left cauda, left epididymis, and left testis were weighed.
Litter observations:
not examined
Postmortem examinations (parental animals):
Complete histopathology was performed on 0 and 400 ppm core study rats and mice. In addition to gross lesions and tissue masses, the following tissues were examined to the no-effect level: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gall bladder (mice), heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung, lymph nodes (mandibular, mesenteric, bronchial, and mediastinal), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. The kidneys from all male rats were also evaluated.
Postmortem examinations (offspring):
not examined
Statistics:
Organ and body weight data were analyzed by parametric multiple comparison procedures (Duunett, 1955; Williams, 1971 and 1972). Hematology, clinical chemistry, urinalysis, renal toxicity, and spermatid and epididymal spermatozoal data, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were examined and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.
Reproductive indices:
not examined
Offspring viability indices:
not examined
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
non-neoplastic – kidney lesions in male rats
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
No mortality was recorded in any of the animals. Mean body weight and body weight gains were similar to sham-exposed animals at study termination. Significant increases in glucose/creatinine, protein/creatinine, AST/creatinine and LDH/creatinine ratio was observed in male rats consistent with renal toxicity data. Mild increases were seen in AST/creatinine and LDH/creatinine ratio in female but did not correlate with the lack of kidney weight increases in females and hence not considered toxicologically relevant.
Transient increase in reticulocyte counts (day 3) and platelet counts (day 23) occurred in a dose-dependent trend in males and females. Changes were however minimal, had disappeared by study termination and were not considered toxicologically relevant.
Transient decreases in serum AP and increases in bile acid concentrations occurred on days 3 and 23 but had returned to control values by study termination. No changes were observed with serum ALT or SDH (other markers of hepatocellular injury) through the duration of the study.
Relative kidney weights and liver weights of male rats exposed to 50 ppm or greater and absolute kidney weights of 200 and 400 ppm males were significantly increased. Liver weights for 200 and 400 ppm females were also increased. Liver weight increases in the absence of pathological correlates were considered physiological adaptations to increased metabolic load.
Kidney lesions noted in males were associated with α2u-globulin nephropathy.

There were no significant differences between exposed and chamber control animals in sperm evaluations in males or vaginal cytology parameters in females.
Dose descriptor:
NOAEC
Effect level:
>= 400 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: 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
Reproductive effects observed:
not specified
Conclusions:
The NOAEC >=400 ppm for male/female for reproductive tissue evaluations and estrous cycle characterization
Executive summary:

Reproductive tissue for males and estrous cycles in females were evaluated following decalin (a C10 cycloparaffin) exposure. Groups of 25 male and 20 female F344/N rats were exposed to 0, 25, 50, 100, 200 or 400 ppm decalin vapor for 6 hrs/day, 5 days/week for 14 weeks. Samples were collected for sperm count, motility and vaginal cytology evaluations on rats exposed to 0, 100, 200 or 400 ppm. Parameters evaluated were spermatid counts per testis, per gram testis, per cauda and per gram cauda and epididymal spermatozoal motility. Left cauda, left epididymis and left testis were weighed. In females, vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from rats and mice for the purpose of vaginal cytology evaluations. Percentage of time spent in various estrous cycle stages and estrous cycle length were evaluated.

No clinical findings were noted to be related to decalin exposure. With respect to reproductive parameters, there was a statistically significant decrease in spermatid head counts (107)/testis in the 400 ppm dose group, however the significance of this finding does not appear to be clear or relevant to decalin exposure since there were no changes in spermatid counts /g testis, /g cauda epididymis or /cauda epididymis when compared with chamber controls. According to the NTP, “the decrease in spermatid heads/testis was not supported by other spermatid and sperm measurements and is considered of no biological significance”. Estrous cycle length of exposed females did not change compared to chamber controls.

Conclusion –Decalin exposure had no effect on sperm parameters of estrous cycle in male and female rats. NOAEC for fertility in both sexes ≥ 400 ppm (highest concentration tested).

Endpoint:
fertility, other
Remarks:
Sub-chronic toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report equivalent or similar to OECD guideline 413
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
other: OECD TG 413
Principles of method if other than guideline:
Male/female rats were exposed to stoddard solvent vapor (0, 138, 275, 550, 1100 or 2200 mg/m3) for 6 hours plus 12 minutes/day, 5 days/week for 14 weeks. Reproductive tissue evaluations for male rats included epididymis, cauda epididymis and testis weights, spermatid measurements and epididymal sperm motility measurements. Female rats were assessed for all 4 stages of the estrous cycle.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic laboratory animals and services, Germantown, NY
- Diet (e.g. ad libitum except during exposure and urine collection periods): NTP-2000, Zeigler Bros., Inc, Gardners, PA
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 13 days (males), 14 days (females)
- Age at study start - 5-6 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 26
- Relative humidity: 55 +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Vapor was generated by pumping substance through a heated glass column filled with glass beads to increase surface area for evaporation. Heated nitrogen, pumped into the column, was used to vaporize the substance before transporting to exposure chamber at elevated temperature to avoid condensation. In exposure room, vapor was mixed with additional heated air in distribution manifold (at constant pressure) before being pumped through individual temeperature-controlled delivery lines to each exposure chamber. Vapor was diluted with conditioned chamber air to achieve desired exposure concentration.

Exposure chambers were designed to maintain a uniform vapor concentration throughout the chamber (total active mixing volume of 1.7 m3/chamber). Particle detector was used in exposure chambers to monitor particle count. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) ensuring that vapor and not aerosol was produced.

Build-up and decay rates for chamber vapor concentration were determined with animals present in chambers. At a chamber airflow rate of 15 air changes/ hour, time required to achieve 90% of target concentration (T90) was determined to be 12 minutes.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Substance concentration in exposure chambers were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately 5 – 6 times during each 6-hour exposure period using a 12-port stream select valve. The on-line gas chromatograph was checked throughout the day for instrument drift against an on-line standard of major n-paraffins of Stoddard solvent IIC. Mean results for decane, undecane and dodecane of 100.1%, 100.0% and 99.9% respectively during 2-year inhalation studies indicated that the composition of Stoddard solvent IIC did not change significantly during exposure.
Duration of treatment / exposure:
6 hours plus T90 (12 minutes)/day
Frequency of treatment:
5 days/week for 14 weeks
Details on study schedule:
Animals were distributed randomly into groups of approximately equal initial mean body weights. Groups of 10 male and 10 female rats were exposed to Stoddard solvent IIC at concentrations of 0, 138, 275, 550, 1,100, or 2,200 mg/m3, 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. Feed was available ad libitum except during exposure periods; water was available ad libitum. Animals were housed individually per cage.
Remarks:
Doses / Concentrations:
0 (control), 138, 275, 550, 1100 or 2200 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals/sex/group
Control animals:
yes, sham-exposed
Parental animals: Observations and examinations:
Observed twice daily; core study animals were weighed initially, weekly, and at the end of the studies; clinical findings were recorded weekly and at the end of the studies.
Oestrous cyclicity (parental animals):
Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from core study females exposed to 0, 550, 1,100, or 2,200 mg/m3 for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Sperm parameters (parental animals):
At the end of the studies, sperm samples were collected from male core study animals in the 0, 550, 1,100, and 2,200 mg/m3 groups for sperm count and motility evaluations. The following parameters were evaluated: spermatid heads per testis, per gram testis, per cauda and per gram cauda, and epididymal spermatozoal motility. The left cauda, left epididymis, and left testis were weighed.
Postmortem examinations (parental animals):
Complete histopathology was performed on 0 and 2,200 mg/m3 core study rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gall bladder (mice), heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung with mainstem bronchi, lymph nodes (mandibular, mesenteric, bronchial, and mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. The larynx, lung, nose, and trachea of rats and mice; kidney of rats; and spleen of female mice were also examined in the remaining exposure groups.
Statistics:
Organ and body weight data were analyzed by parametric multiple comparison procedures (Duunett, 1955; Williams, 1971 and 1972). Hematology, clinical chemistry, urinalysis, renal toxicity, and spermatid and epididymal spermatozoal data, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were examined and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
non-neoplastic – kidney lesions in male rats
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
No mortality was recorded in any of the animals. Mean body weight and body weight gains were similar to sham-exposed animals at study termination. Final mean body weight of 275 mg/m3 females was greater than that of chamber controls but with no toxicological significance. Exposure-related decreases were noted for ALT in males and females. These changes however were not considered adverse. Minimal decreases in hematocrit values, hemoglobin concentration and erythrocyte counts were noted at study termination in 2200 mg/m3 males. This decrease was however minimal and not considered of toxicological relevance. Relative liver, kidney and testis weights of all exposed group males and absolute kidney weights in 550 mg/m3 or greater exposed males were increased. Increased relative testis weights did not appear to be toxicologically relevant since absolute weights were not increased. While the liver weights may have been due to an adaptive response since it lacked any pathological correlates, increased relative and absolute kidney weights correlated with the observation of α2u-globulin nephropathy in all exposed males.
Gross observations of kidney at necropsy revealed increased incidence of hyaline droplet accumulation, regeneration of tubular epithelium and increased severity of granular casts.
Incidences of goblet cell hypertrophy of the nasal respiratory epithelium in 2200 mg/m3 males and 1100 mg/m3 females were increased.
Dose descriptor:
NOAEC
Effect level:
>= 2 200 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: 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
Reproductive effects observed:
not specified
Conclusions:
NOAEC for fertility in rats ≥ 2200 mg/m3.
Executive summary:

Groups of 10 males and 10 female F344/N rats were exposed to Stoddard solvent (C9-C14 aliphatics; 25% aromatics) via inhalation at concentrations of 0, 138, 275, 550, 1100 or 2200 mg/m3, 6 hrs (plus an additional 12 minutes)/day, 5 days/week for 14 weeks. At the end of the 14-week exposure, all animals were sacrificed and necropsies performed on all study animals. Heart, right kidney, liver, lung, right testis and thymus were weighed. Most importantly for reproductive tissue and oestrus cycle evaluations, sperm samples were collected from study animals in the 0, 550, 1100 and 22000 mg/m3 groups for sperm count and motility evaluations. Parameters evaluated were number of spermatid heads/testis, per gram testis, per cauda and per gram cauda and epididymal spermatozoal motility. Left cauda, left epididymis and left testis were weighed. In females, vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from rats exposed to 0, 550, 1100 and 22000 mg/m3 for the purpose of vaginal cytology evaluations. Percentage of time spent in various estrous cycle stages and estrous cycle length were evaluated.

Absolute tissue weights of gonads for male rats and mice did not change with exposure. The only adverse effects noted in the 3-month study were epididymal sperm motility decreases in male rats exposed to Stoddard solvent from 550 mg/m3 up to highest dose. It is important to note that the changes in epididymal sperm motility occurred with no positive dose-response trend. Interestingly, the changes in sperm motility occurred absent of any morphological or microscopic effects on the testes in the same doses over a 2-year exposure period in both species. As indicated by the NTP, despite the slight statistically significant changes in sperm motility, these changes do not appear to be relevant to fertility. In support of this conclusion, Chapin et al., (1997) evaluated 80 different multi-generational mouse studies covering 72 chemicals (33 chemicals had adverse reproductive effects in males and females, 7 affected females and pups only, 2 chemicals affected males only while 17 where negative) to determine the relationships between various necropsy endpoints evaluated in males and females and functional indicators of reproduction. In males specifically, fertility was significantly reduced when > 15% sperm abnormalities was observed. Epididymal sperm motility affected fertility only when percent motility was ≤ 37%. No sperm abnormalities were observed with rats exposed to Stoddard solvent and % motility was much greater than the 40% threshold identified by Chapin et al (1997). For females on the other hand, Stoddard solvent had no effect on estrous cycle.

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
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.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
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 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).
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.
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.

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
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.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
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 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.
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 3 000 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No 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:
no effects observed
Gross pathological findings:
not examined
Histopathological findings:
not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 3 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse treatment-related effects observed at the highest dose tested.
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.

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report equivalent or similar to OECD guideline 415: GLP
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Lakeview, NJ
- Diet: Purina Certified Rodent Chow #5002 (Checkers)
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 3 weeks
- Age at study start - 7 week

Prior to parturition, cages housing presumed-pregnant animals were fitted with a stainless steel floor. Dams were provided with Beta Chip Bedding (Fisher and Sons, Bound Brook, NJ) as nesting material.
Route of administration:
dermal
Details on exposure:
Rats were clipped before the first day of study and weekly thereafter. Stock 461 was drawn up into a 1 cc syringe (calibrated in 0.01 cc) and during dispensing was spread evenly on the clipped dorsal skin of the rat using the tip of the syringe without the use of a needle. Application sites were not covered. Amount of test material applied to each animal was calculated using the most recently recorded body weight for each animal, the dose level and the density of the test material. Rats were fitted with cardboard Elizabethan-style collars to minimize ingestion of the test material. Collars were lined with soft rubbier tubing to minimize development of irritation or lesions.

During the mating period, the test material remained on the animals for a minimum of four hours. Excess material was removed with a gauze pad from the application site of each animal prior to cohabitation in an attempt to minimize test material ingestion during preening.

Untreated controls were not clipped nor collared and received no treatment. Dermal control female rats were clipped and collared as above. Dorsal skin of each rat was stroked with the tip of a 1 cc syringe but no test material was applied.


Details on mating procedure:
During the mating period, female rats which had not previously borne pups were placed with adult male rats from their corresponding treatment group in a ratio of 1:1 and observed daily for evidence of having engaged in breeding activity. Each morning during the period of cohabitation, the drop-pan papers under the animal cages were checked for the presence of expelled vaginal sperm plugs; additionally, each female rat was examined for the presence of in situ vaginal sperm plugs. Vaginal lavage fluid was obtained from each female which exhibited a vaginal sperm plug in situ or on the drop-pan papers, and was examined for the presence of spermatozoa. Females that were positive for sperm plug as well as for spermatozoa were considered to be at day 0 of presumed gestation and were placed in individual housing units. The cohabitation was continued until 20 presumed-pregnant female rats/group were obtained.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Premating period - 10 weeks
Mating period - 3 weeks
Postmating period - Gestation days 0-20
Frequency of treatment:
5 days/week
Details on study schedule:
Female rats were randomly divided into 5 groups (untreated control, dermal control, 125, 500 or 2000 mg/kg bw) using computer-generated random numbers. Each group consisted of 20 rats dosed dermally from premating to gestation day 20. 10 additional females were administered the test material during the premating period to assure the obtainment of twenty presumed-pregnant females.
Remarks:
Doses / Concentrations:
0, 125, 500 or 2000 mg/kg body weight
Basis:
other: Nominal dose calculated on the basis of animal body weight, dose level and density of test substance
No. of animals per sex per dose:
20 female rats/dose
Control animals:
yes, concurrent no treatment
yes, sham-exposed
Parental animals: Observations and examinations:
All rats were monitored throughout the study period until sacrifice for changes in appearance, behavior, excretory function, signs of ill-health and mortality. Additionally, each presumed-pregnant female was monitored during gestation for signs of abortion or premature delivery. During parturition, dams were observed for signs of dystocia (difficult delivery), and during lactation for maternal behavior (pup retrieval and nursing the litter); only observations that warranted comment were recorded.

Body weight of each rat was measured to the nearest 0.1 gram once weekly during the premating phase. No body weights were measured during the mating period. During the postmating phase, the body weight of each presumed-pregnant female was measured to the nearest 0.1 gram on days 0, 3, 6, 10, 13, 16, 18 and 20 of gestation and on days 0, 4, 7, 10, 14 and 21 of the postpartum period (lactation). The amount of food consumed for each animal was calculated for each week of the premating period and for gestation day intervals 0-3, 3-6, 6-10, 10-13, 13-16, 16-18 and 18-20. Food consumption was not measured during the mating and postpartum periods.
Oestrous cyclicity (parental animals):
The state of the estrous cycle of five female rats from groups I (untreated control), II (Dermal control) and V (2000 mg/kg bw/day) was determined daily (5 days/week) for two weeks prior to the mating period and continued until evidence of engaging in breeding activity was obtained. Each morning, vaginal lavage fluid was obtained from each female. A drop of the lavage fluid was examined under a microscope and the stage of estrus recorded.
Litter observations:
All offspring were observed throughout the postpartum period until sacrifice for changes in appearance, behavior, body weight, signs of nursing (milk in the stomach), ill-health or mortality. Additionally, as soon after birth as possible, all viable neonates were sexed and examined for external anomalies. Subsequently, each pup was monitored for opening of the eyes (eyelid disjunction) and the ability to right itself when placed on its back. Pups were weaned on postpartum day 21.
Postmortem examinations (parental animals):
Each female rat which delivered was sacrificed by over-exposure to carbon dioxide gas on postpartum day 21 or as soon as time permitted. Dams whose litters died during the postpartum period were sacrificed as soon as the operating schedule permitted. In all instances, the thoracic and abdominal cavities were exposed and all organs were examined grossly for evidence of parthosis. Reproductive organs (ovaries and uterus) were removed, examined grossly, weighed and fixed in 10% neutral buffered formalin. The number of uterine implantation sites in each urerine horn was counted and recorded. All other remarkable findings were recorded.
Postmortem examinations (offspring):
Surviving weanlings (from postpartum day 21) were sacrificed by over-exposure to ether or carbon dioxide gas on postpartum day 28 or as soon thereafter as time permitted. thoracic and abdominal cavities of each weanling were exposed and all the organs were examined grossly for evidence of pathosis.
Statistics:
Data generated during the gestation and postpartum phases wre analyzed by analysis of variance followed by group comparisons using the Fisher's exact or Dunnett's test. Differences were considered statistically significant if the probability of the difference being due to chance was less than 5% (p < 0.05).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dermal irritation, scabs and flaking of skin
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:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Dermal irritation was observed at the site of application which persisted after administration of test material. There was no treatment-related effect in maternal parameters such as food consumption, body weight gain. Reproductive parameters such as length of gestation, number of implants, litter size did not change compared to chamber controls.
Dose descriptor:
NOAEL
Effect level:
>= 2 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: There were no adverse effects to reproductive performance.
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
Offspring body weights, viability, eyelid disjunction and surface righting ability were not affected adversely by in utero exposure to stock 461
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse treatment-related effects observed at the highest dose tested.
Critical effects observed:
no
Reproductive effects observed:
not specified
Conclusions:
NOAEL for fertility was greater than or equal to 2000 mg/kg body weight (highest dose tested)
Executive summary:

A one-generational reproductive toxicity study was conducted to evaluate the reproductive performance (gonadal function, all 4 stages of the estrus cycle, fertilization, implantation of the egg, gestation period and parturition) of a C16-C30 highly purified light mineral oil. The pre-natal developmental phase also evaluated pup growth and development up to weaning. Female Sprague-Dawley rats were dermally exposed to test material for 10 weeks premating period (5 days/week), a 3-week mating period (5 days/week) and up to gestation day 20. Test material was applied once daily to clipped, intact dorsal skin at a dose level of 125, 500, 2000 mg/kg bw/day. The application sites were not occluded and evaporation was not expected to affect dosing due to low vapor pressure. Treatment-related dermal irritation (erythema, formation of scabs, skin flaking) at site of exposure were observed in almost all exposed rats. Neonatal deaths observed 0-4 days postpartum was not considered treatment-related as this was also observed in the control groups. No clinical signs of systemic toxicity were observed and there were no effects on maternal body weight and food consumption. No differences were observed in mean numbers of implantation sites, live pups/litter, live birth index (total pups born alive/total pups born) either at birth, days 4 or 21 postpartum. There was no change in mean pup body weights and no pathological observations were seen in necropsied organs of the pups. NOAEL for mineral oil was ≥ 2000 mg/kg bw/day for fertility and pre-natal development.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report equivalent or similar to OECD guideline 416
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Source - Charles River Japan
Feed - Ad libitum (NIH-07M; CLEA Japan)
Water - ad libitum
Age at study initiation - F0 (5 weeks), F1 (3 weeks)

Temperature - 24 +/- 2 deg C
Humidity - 55 +/- 10%
Light/dark cycle - 12/12 hrs
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
Oral administration was done by gavage using Teflon gastic tubes. n-butylbenzene was disolved in olive oil and dose volume adjusted to 5 ml/kg. Controls were dosed with an equal volume of olive oil only.
Details on mating procedure:
Males and females were paired 1:1 at 15 weeks of age (10 weeks from dosing start in F0 parental animals or at 13-15 weeks of age; 10-12 weeks from dosing start at 3 weeks of age in F1 parental animals). Paired females were examined on the next morning for the presence of vaginal plugs or sperm in vaginal smears. When either was observed, it was judged that copulation had taken place and the day was identified as gestation day 0.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
F0 parental animals
Males - Approximately 16 weeks: includes 10 weeks before mating from 5 weeks old, 2 weeks mating period and until day of autopsy
Females - Approximately 14-16 weeks: Includes 10 weeks before mating from 5 weeks old, 2 weeks mating period, gestation period and up til gestation day 21

F1 parental animals
Males - Approximately 18 weeks: including 10 weeks before mating from 3 weeks of age (at time of weaning), 2 weeks mating period and up till one day prior to autopsy
Females - Approximately 19-21 weeks: including 10 weeks before mating from 3 weeks of age at time of weaning, 2 weeks mating period, gestation period and up till gestation day 21 after delivery
Frequency of treatment:
Daily
Details on study schedule:
Rats were randomly distributed into 4 dose groups
Remarks:
Doses / Concentrations:
0, 30, 100 or 300 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
24/sex/group
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Individual animals observed twice daily before and after administration of test substance for survival, death, external appearance or behavior.
Body weights and food consumption for males measured weekly
Body weights of females measured once a week during maturation period and on gestation days 0, 7, 14 and 20 and pst natal days 0, 4, 7, 14 and 21. Food consumption of females was measured once a week during maturation period and on GDs 1, 7, 14, 20 and on PNDs 1, 4, 7, 14 and 21.
Oestrous cyclicity (parental animals):
Estrus count, estrous interval were evaluated
Sperm parameters (parental animals):
Sperm (from caudal part of right epididymis) was collected from 10 animals in each group and evaluated for sperm count, sperm motility and morphology.
Litter observations:
Litter were observed for clinical signs daily during the lactation period. Body weights were measured on PNDs 0, 4, 7, 14 and 21. Anogenital distance was measured on PND 4 for F1 offspring only. To detect developmental effects, completion rates for littermates with regard to pinna detachment (PND 4 to completion), Incisor eruption (PND 10 to completion) and eyelid separation (PND 15 to completion) was determined. Righting reflex and ipsilateral flexor reflex (PND 5 to completion), visual placing reflex (PND 16 to completion) and preyer's reflex (from PND 28 to completion) was used to detect effects on development related to endocrine disruption.
Postmortem examinations (parental animals):
Weights of brain, pituitary gland, thyroid, thymus, liver, kidneys, spleen, adrenal glands, testes, epididymis, seminal vesiglces, prostate, ovaries and uterus in male and female F0 and F1 parental animal were evaluated. All organs including oviduct, uterus, uterine, uterine cervical part, vagina, mammary glands and macroscopically abnormal sites were evaluated in paraffin embedded sections stained with hematoxylin and eosin.
Postmortem examinations (offspring):
Weights of the brain, thymus, spleen, testes, epididymis, ovaries and uterus in male and female F1 and F2 weanlings in all litters were evaluated.
Statistics:
Mean values and standard deviations for data on body weights, food consumption, number of days needed for copulation, gestation length, organ weights, estrous counts, estrous cycle length, anogenital distance, hormone levels, sperm count, implantation count, litter size and number of offspring born were determined for each group. Variance homogeneity was tested by Bartlett's method. Group comparison with control group was made using a Dunnett's multiple-comparison test when the variance was homogenous among groups. For non-homogenous variance, comparison was conducted with Steel's multiple comparison test.

Comparisons between controls and single dose groups for gestation index, mating index, fertility index and sex ratio was assessed by Chi-square test. For still birth rate, birth rate, viability, weaning index and the results of physical development testing and reflex response testing, rate for externally abnormal fetus, sperm motility and rate of sperm abnormality, the Wilcoxon;s rank sum test was used.
For the results of histopathological exams, the Mann-Whitney's U test was used.
Levels of significance was concluded at the 1 and 5% level in all tests.
Reproductive indices:
Mating index
fertility index
gestation rate
litter size
Offspring viability indices:
Sex ratio
birth rate (%)
Viability on PND 4
Weaning rate
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain inhibited during nursing period for F0 and F1 parental animals. No effect in males
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain inhibited during nursing period for F0 and F1 parental animals. No effect in males
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:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Increased liver weights were noted in animals recieving 30, 100 and 300 mg/kg/day of test substance which was associated with centrilobular hepatocytic hypertrophy in the highest dose groups for F0 and F1 parental animals. This was considered not relevant since this is a physiological adaptation (hyperplasia of the smooth ER) to chronic cyt p450 drug-metabolizing enzyme induction to aromatic substances. Increased kidney weights was noted in F0 and F1 parental animals at the 100 and 300 mg/kg/day groups. Deposition of hyaline droplets in proximal tubules noted in F0 males at the same doses suggested this was not relevant to humans and is associated with male rat-specific alpha-2-u globulin nephropathy.
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No adverse effects on fertility for parental F0 and F1 animals
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Thymus weight increases were noted in F1 males and females and F2 females all at the 300 mg/kg/day dose level
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Increased thymus weights in F1 offspring males and females
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No adverse effects
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Increased relative thymus weights in F2 females
Reproductive effects observed:
not specified
Conclusions:
NOAEL was determined to be >=300 mg/kg bw/day for fertility. NOAEL was determined to be 100 mg/kg/day for developmental effects due to increased thymus weights noted in males/females in the F1 offspring and female offspring of the F2 generation.
Executive summary:

N-butylbenzene, a C10 aromatic hydrocarbon, was examined for toxicity in a two-generation reproductive toxicity study. N-butylbenzene was administered by oral gavage at dose levels of 0, 30, 100, and 300 mg/kg/day to groups of Crj: CD (SD) IGS rats (24 males and 24 females per group) over 2 generations, and the effects on fertility of the parental animals and development/growth of the offspring were investigated. It was concluded that n- butylbenzene did not induce reproductive toxicity in the F1 parental animals and no effects on the endocrine system were observed. Therefore, the NOAEL was determined to be >=300 mg/kg bw/day. Based on this study, C10-C12 Aromatic Hydrocarbons are not expected to be reproductive toxicants.

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 422:GLP.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Number of Animals: Males, 48; females, 48 (total)
Crj:CD (SD strain) SPF male and female rats 8 weeks old were purchased from Nippon Charles River Co., Ltd., and quarantined for 14 days. The administration was started when the animals were 10 weeks old, and the mean body weight (body weight range) of males at the start of administration was 400.3 g (374-431 g), and that of females was 226.5 g (192-255 g).

The animals were individually placed in metal bracket cages with a metal mesh floor before mating, one male and one female per cage during mating, one mother animal per cage during gestation, and a mother and her newborns after birth.

Animals were maintained in a barrier-isolated room with a temperature of 23 +/- 3°C, humidity of 55 +/- 10%, ventilation changes of 10-15 times per hour, and an illumination of 12 hr/day. After the 17th day of gestation, the metal mesh floor was changed to a stainless steel pan spread with a floor-covering material for experimental animals. Solid feed (CRF-1, manufactured by Oriental Yeast Industry Co., Ltd.), and tap water (tap water of the City of Sapporo) were given freely.
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
The administration of the test substance was carried out by oral gavage. The volume administered was 5 mL per kg of body weight, and it was calculated based on the results of body weight measured on the day closest to the day of administration. The administration period was 46 days, which including 14 days before mating and during the mating period for males. The administration period for the female rats began 14 days before mating and continued until after the first 3 days of nursing. The administration was started when the animals were 10 weeks old, and the mean body weight (body weight range) of males at the start of administration was 400.3 g (374-431 g), and that of females was 226.5 g (192-255 g).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The administration period was 46 days, which including 14 days before mating and during the mating period for males. The administration period for the female rats began 14 days before mating and continued until after the first 3 days of nursing.
Frequency of treatment:
once per day
Remarks:
Doses / Concentrations:
0 (vehicle), 100, 300, 1000 mg/kg/day
Basis:
actual ingested
oral gavage
No. of animals per sex per dose:
Males, 12 ; females, 12 per dose
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
General conditions, body weight and feed intake:
For all cases, the general conditions were observed once a day throughout the study term. The body weight measurements were carried out on the 1st (before administration), 2nd, 5th, 7th, 10th and 14th day after administration and subsequently, every 7 days (including the last day of administration) as well as 0th, 1st, 3rd, 5th, 7th, 10th, 14th, 17th and 20th day of gestation and the 0th, 1st and 4th day of nursing for females. In addition, the body weight gain and rate were calculated from the 1st day of administration to the 46th day for males and for females, from the 1st day to the 14th day, 0th day to the 20th day of gestation and 0th day to the 4th day of nursing. The amount of feed intake was measured on the same days as those of body weight measurements except mating period and dissection day for males and 0th day of gestation and 0th day of nursing for females. Incidentally, with respect to the number of day of pregnancy, the successful copulation day was set as the 0th day of gestation, and in the case of lactation, the day of delivery completion was set as the 0th day of nursing.

Urinary tests:
In the final week (43rd-44th day of administration) during the administration term, 6 male cases of each group were placed in metabolism-measurement cages, and their urine samples were collected under non-starvation conditions. For urine samples, collected in about 3 hr, pH, protein, glucose, ketone, urobilinogen, bilirubin, occult blood reaction, and sedimentation (microscopic observation) were tested, and for urine samples collected for 21 hr, volume, and specific gravity were measured. In addition, the amount of drinking water (weight) in urine samples was also measured.

Hematological tests:
Before dissection, all male animals were starved for about 16 hr, blood samples were collected from the femoral vein under ether anesthesia, and EDTA•2K-treated blood samples were used to measure the erythrocyte count, mean erythrocyte volume, platelet count, leucocyte count, hemoglobin content (cyanmethemoglobin method), hematocrit (calculated from erythrocyte count and mean erythrocyte volume), mean erythrocyte hemoglobin content (calculated from erythrocyte count and hemoglobin content), mean erythrocyte hemoglobin concentration (calculated from hematocrit and hemoglobin content), reticulocyte proportion (Brecher method) and leucocyte fraction (microscopic observation). In addition, untreated blood samples were used to measure coagulation time (fluid viscosity change air pressure measurement, Gryner Microcogulometer). Furthermore, plasma samples, which were prepared by collecting blood samples from the abdominal aorta, treating with sodium citrate and subsequently carrying out centrifugation at 3,000 rpm for 10 min, were used to measure prothrombin time (thromboplastin method) and activated partial thromboplastin time (ellagic acid method).

Hematobiochemical test:
After the hematological tests, serum samples of all male cases, which were prepared by collecting from the abdominal aorta, were used to measure GOT, GPT (IFCC method), GPT (glutamyl-p-nitroanilide substrate clathrate method), choline esterase (butyrylthiocholine iodide substrate method), blood glucose (hexokinase method), total cholesterol and phospholipids (enzymatic method), triglyceride (free glycerol deduction method), total bilirubin (azobilirubin method), urea nitrogen (urease-indophenol method), creatinine (Yaffe method), calcium (OCPC method), inorganic phosphorus (Fiske-Subbarow method), total protein (Biuret method) and albumin (BCG method) (Hitachi automated analyzer, Model 7150); sodium and potassium (flame photometry: Corning flame photometer, Model 480); chlorine (coulometric titration method: Hiranuma chloride counter, Model CL-6M); A/G ratio (calculated from total protein and albumin); and protein fraction (cellulose acetate electrophoresis).
Litter observations:
The viability of the newborns was confirmed once a day from delivery completion to the 4th day of nursing. The viability of newborns for 4 days [(No. of live pups on the 4th day/No. of pups born) x 100] was calculated. In addition, the general condition and appearance of the pups was observed. The body weight was measured on the 0th, 1st and 4th day of nursing, and the body weight gain and the rate of body weight gain were calculated. Dead pups were dissected immediately after discovery. All pups were sacrificed and dissected on the 4th day of nursing.
Postmortem examinations (parental animals):
Dissection and organ weight measurement:
After the 46th day of administration, all males were sacrified ex sanguine under ether anesthesia after blood sampling and dissected. Any newborns that died were dissected immediately after discovery. Females that successfully copulated were sacrificed on the 4th day of nursing. Female rats who did not successfully copulate on the 25th day of gestation (infertile cases) on the 26th day of gestation were sacrified. The implantation sites in the uterus and corpus luteum of pregnancy in the ovaries were counted. In addition, the weight measurements were carried out for the liver, kidney, thymus gland, adrenal gland, testes, epididymis and ovary, and the ratio to body weight was calculated.

Histopathological observation:
The following tissues were embedded in paraffin and stained with hematoxylin-eosin or with oil red O staining/ luxol fast blue-Bodian double stain to conduct a histopathological examination: the liver, kidney, spleen, heart, lung, brain (cerebrum and cerebellum), hypophysis, thymus gland, adrenal gland, thyroid gland, stomach (anterior stomach and glandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, testes, epididymis, prostate gland, ovary and abnormal sites.
Statistics:
Fisher’s accuracy probability test was carried out to compare the control and undecane-administered groups for the sexual cycle, copulation index, fertility index, gestation index and nursing index. Other test parameters were analyzed by using Bartlett’s homogeneity of variance and subsequently single dimensional configuration variance analysis or Kruskal-Walls method. If the results were found to be significant, the Dunnett ‘s method or Mann-Whitney U-test method was used to compare the undecane-administered groups from the control group. However, those qualitative items in urinary tests were analyzed by using the Kruskal-Walls method and Mann-Whitney U-test method. The viability of newborns on the 4th day and body weight were analyzed using the litter as a unit and the results were compared with the control group. A significance level of less than 5% was considered to be statistically significant.
Reproductive indices:
Reproductive ability test:
For female rats, vaginal smear specimens were prepared every day starting from 10 days before administration. Specimens were prepared until successful copulation was confirmed in order to evaluate any abnormalities in the sexual cycle.

On the 14th day of administration, males and females rats (1 to 1 pairs; same dose) were allowed to cohabit for a maximum of 14 days. Successful copulation was confirmed when spermatozoa were detected in the vaginal smear of the female. Gestation was confirmed through the detection of implantation sites in the uterus. In addition, the copulation index [(No. of pairs with successful copulation/No. of pairs mated) x 100] and fertility index [(No. of pregnant animals/No. of pairs with successful copulation) x 100] were calculated.
Offspring viability indices:
The viability of the newborns was confirmed once a day from the delivery completion day to the 4th day of nursing. The viability of newborns for 4 days [(No. of live pups on the 4th day/No. of pups born) x 100] was calculated. In addition, the general condition and appearance of the pups was observed. The body weight was measured on the 0th, 1st and 4th day of nursing, and the body weight gain and the rate of body weight gain were calculated. Dead pups were dissected immediately after discovery. All pups were sacrificed and dissected on the 4th day of nursing.
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
In the repeat dose toxicity test, salivation was observed in males and females given 300 and 1000 mg/kg. Body weight gain was suppressed in males given 1000 mg/kg, and body weights were increased in females given 1000 mg/kg during the lactation period. Food consumption was decreased in males given 300 and 1000 mg/kg in the first half of the administration period, increased in males given 1000 mg/kg in the second half of the administration period, and increased in females given 1000 mg/kg in the second half of pregnancy and during the lactation period. Hematological and blood chemical examinations revealed a decrease in hemoglobin concentration, an increase in the white blood cell count, a decrease in albumin, and increases in a2-globulin, GPT, cholinesterase and total cholesterol in males given 1000 mg/kg. Relative liver weights and absolute and relative thymus weights were increased in males given 1000 mg/kg, and absolute and relative liver weights were elevated in females given 1000 mg/kg. No effects were detected in the autopsy or histopathology findings. Due to the lack of effects in both sexes and the lack of corresponding histopathology, these effects are determined not to be toxicologically relevant. The NOAEL for repeat dose toxicity is considered to be 1000 mg/kg/day for both sexes.

Reproductive and developmental toxicity
No effects of undecane administration were observed on the sex cycle of females and copulation and conception of males and females. In addition, no effects of undecane administration were observed on the weights of reproductive organs (testis, epididymis and ovary) and there were no abnormalities noted in the dissection and histopathological examination. Incidentally, the reproductive organs of infertile cases showed no histopathological findings suggesting the causes. Abnormal cases observed in this study were confirmed to be spontaneous when compared to historical controls. Those cases observed in the present study were considered to be unrelated to undecane.

The number of live or dead pups delivered per each dam in the undecane-administered group showed no apparent difference from that in the control group if those dead and unknown pups in those cases of abnormal delivery or death of all of the litter during nursing were excluded. The body weight gain rates of both males and females in the 1,000 mg/kg group were observed to be reduced, but no effects of undecane were observed as a result of general condition observation or dissection. The NOAEL for reproductive performance is considered to be 1000 mg/kg/day.
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects noted at highest dose tested.
Dose descriptor:
NOAEL
Remarks:
repeat dose
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects noted at highest dose tested.
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:
not examined
Histopathological findings:
not examined
No effects were detected with regard to reproductive ability, reproductive organ weights, or autopsy or histopathology findings in either sex, and there was no apparent influence on delivery or maternal behavior of dams. Body weight gain was decreased in male and female offspring of the 1000 mg/kg group. This was not considered a toxicological effect. No effects were noted in terms of viability, general condition or autopsy findings of offspring.
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects noted at highest dose tested.
Reproductive effects observed:
not specified
Conclusions:
The NOAEL for repeat dose toxicity is considered to be >=1000 mg/kg/day for both sexes. The NOAELs for reproductive performance is considered to be >=1000 mg/kg/day.
Executive summary:

In the repeat dose toxicity test, males and female rats were given 0, 100, 300 and 1000 mg/kg. Male rats were dosed for 46 days (14 days prior to mating and then during the mating period) and female rat from 14 days before mating to day 3 of lactation. The NOAEL for repeat dose toxicity is considered to be >=1000 mg/kg/day for both sexes. The NOAEL for reproductive performance is considered to be >=1000 mg/kg/day.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 422:GLP
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
not specified
Details on exposure:
Males were treated from day 14 prior to the mating phase until the end of the mating phase and then killed, Females were treated from day 14 prior to mating, through day 4 of lactation and then killed.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Males were dosed from the 14th day prior to mating, during mating until the end of the mating period. Females were dosed from the 14th day prior to the start of the mating phase to day 4 of lactation.
Frequency of treatment:
Single daily dose 7days/week
Remarks:
Doses / Concentrations:
0, 25, 150, or 1000 mg/kg/day (10 ml/kg dosing volume)
Basis:
other: gavage
No. of animals per sex per dose:
10 male, 10 female per group
Control group: 10 male, 10 female, 0.5% methylcellulose
Control animals:
yes
Parental animals: Observations and examinations:
Effects on general toxicity, neurobehavioral activity, clinical chemistry, and hematology were evaluated. Gross necropsies and histopathologic examination of tissues were conducted with emphasis on the male reproductive tract.
Reproductive assessment included mating, conception and fertility indices, reproductive organ weights and gross and histologic examination of the reproductive tract (special emphasis on stages of spermatogenesis in male gonads and interstitial testicular cell structure).
Sperm parameters (parental animals):
stages of spermatogenesis in male gonads and interstitial testicular cell structure
Litter observations:
Developmental toxicity assessment included, observations of external abnormalities, number of live and still births, mortality, sex determination and weights of pups.
Statistics:
Adult body weights and feed consumption, maternal body weight gains, gestation length and pup body weights were analyzed by ANOVA. Mean mating time was analyzed via the Kaplan Meier method. Pregnancy rates and mating, conception, viability index, post implantation losses, fertility and gestation indices were analyzed by the trend test, Chi-square 2XN and Fisher's exact test (all one tailed). The probability of survival per group was calculated by the product-limit procedure of Kaplan-Meier. Both a trend test and a log-rank test were used to analyze differences in survival among groups.
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 specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
No deaths or clinical signs of toxicity or behavioral changes were noted. No significant differences in body weights or feed consumption were observed. Startle reflex, open field test, and forelimb grip reflex performance data also revealed no treatment-related findings in the parental animals. There were also no treatment-related changes in hematology or blood chemistry parameters, organ weights or gross pathology. An apparent treatment-related, slight to moderate hyperplasia of the non-glandular mucosa of the stomach, associated with degeneration, hyperkeratosis and submucosal subacute inflammation and, in a few cases, with erosion, was seen in animals of all treated groups. This effect was considered an artifact of the dosing method and not directly related to the toxicity of the test material. No other treatment related histological changes were observed.

There were no treatment-related effects at any dose level on any of the reproductive parameters evaluated in this study. These included measures of reproductive performance (mating, conception, gestation length, litter size), offspring survival (gestation and postnatal survival indices, percent pre- and post-implantation loss). The mean mating time of the 1000 mg/kg/day groups was slightly longer than of the control, however, the increase was not statistically significant and within the normal range of variability for this strain of rats. There was a, non dose-related, decrease in fertility (decreased fertility index) was observed in all treated groups (not statistically significant) compared to controls. However, this effect took place in the absence of any adverse effects on reproductive organs and may have resulted from changes in mating behavior due related to stomach irritation experienced by the treated animals.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No effects noted at highest dose tested.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
There were no treatment-related effects at any dose level on any of the reproductive parameters evaluated in this study including offspring survival (gestation and postnatal survival indices, percent pre- and post-implantation loss), pup body weight and pup sex ratio. There were also no treatment-related effects on any of the developmental parameters evaluated including external abnormalities, number of live and still births, mortality, sex determination and weights of pups.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No effects noted at highest dose tested.
Reproductive effects observed:
not specified
Conclusions:
Oral dosing of Linpar 10 to male and female Sprague Dawley rats at levels of 0, 25, 150, or 1000 mg/kg body weight /day produced no evidence of developmental toxicity or teratogenicity and no statistically significant treatment-related effects on any of the reproductive parameters evaluated in this study. Based on these data, the no-observable-adverse effect level (NOAEL) for developmental toxicity was 1000 mg/kg/day and the NOAEL for reproductive toxicity was 1000 mg/kg/day, the highest dose tested.
Executive summary:

Groups of 10 male and 10 female Sprague Dawley rats were dosed with Linpar 10 daily by gavage at exposure levels of 0, 25, 150, or 1000 mg/kg/day Males were dosed from the 14th day prior to mating, during mating until the end of the mating period. Females were dosed from the 14th day prior to the start of the mating phase to day 4 of lactation.  There were no treatment-related effects at any dose level on any of the reproductive parameters evaluated in this study.  These included measures of reproductive performance (mating, conception, gestation length, litter size), offspring survival (gestation and postnatal survival indices, percent pre- and post-implantation loss), pup body weight and pup sex ratio. There were no treatment-related effects at any dose level on any of the developmental paramters evaluated in this study including external abnormalities of pups, number of live and still births, mortality, sex determination, and weights of pups.  Based on these data, the no-observable-adverse-effect level (NOAEL) for developmental toxicity was 1000 mg/kg/day and the NOAEL for reproductive toxicity was 1000 mg/kg/day.

Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study. Minor details not included (for example rat strain)
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Study was extended to the 3rd generation
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
Source - Charles River Labs, Portage, MI
Age on receipt - 6 weeks
Acclimation period - 2 or 3 weeks
Feed - Purina certified rodent chow #5002 (except during exposure)
Water - Ad libitum
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
All animals were exposed in 16 cubic meter glass and stainless steel chambers. Chamber ventilation was provided by an HVAC system separate from the general laboratory air handling system. This air was particulate-filtered and controlled for temperature and humidity. Chamber air flow rate, temperature, and relative humidity were monitored every half-hour during exposure periods.

To generate test atmospheres, nitrogen was heated to 200 deg C by passage through a 1-liter stainless steel cylinder fitted with a 1500-watt band heater and was then introduced at the bottom of a glass column 7.6 cm in diameter and 30 cm long, packed with glass beads. The liquid test material was delivered by a fluid metering pump from a stainless steel safety can, through Teflon tubing, to the bottom quarter of the column. The test sample was vaporized as it flowed up the column with the nitrogen. The vapors were passed to the chamber inlet where dilution with chamber ventilation air reduced the concentration to the desired exposure levels.
Details on mating procedure:
Males and females were cohoused (1:1) for a two-week mating period. Mating was confirmed on visual observation of a vaginal plug.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure levels were monitored with a gas-phase infrared spectrometer. Measurements were made on an hourly basis throughout the study. Accuracy of this method was confirmed by the use of vapor standards. Additionally, the composition (on a weight percentage basis) of the test material within each exposure chamber was determined by gas chromatography during the first exposure week.
Duration of treatment / exposure:
F0 generation
Male and female rats were exposed for a 10-week pre-mating period, a 2-week mating period. Once mating was confirmed, females were further exposed from GD0-20.

F1 generation
Exposure was initiated immediately after weaning period (5-7 weeks of age). Male and female rats were exposed for a 10-week pre-mating period, a 2-week mating period. Once mating was confirmed, females were further exposed from GD0-20.

F2 generation
Exposure in F2 litters commenced immediately following weaning (PND 22) and proceeded as performed in the parental and F1 generations.
Frequency of treatment:
6 hours/day, 5 days/week
Details on study schedule:
F0 generation
Male and female rats were randomly assigned to one of four treatment groups each containing 30 animals/sex/group.

F1 generation
F1 litters were culled to 8 on lactation day 4 (LD 4) but kept with their dams until the end of the weaning period (LD 21). Pups were counted, sexed and weighed individually and weights recorded by sex. Exposure of randomly selected animals (30/sex/group) was performed a week after the weaning period.

F2 generation
F2 litters were treated as described for the F1 generation. Since exposure was initiated immediately after weaning and due to concern for toxicity, 40 animals/sex/group were randomly selected with the intention of reducing this number to 30 by random selection at time of mating. This was doine in the control, 100 and 500 ppm groups. Majority of pups in the 1500 ppm group died during first week of exposure. Survivors were mated after 10 weeks of exposure.
Remarks:
Doses / Concentrations:
100 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
500 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1500 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
30/sex/group for F0 and F1 generations
40/sex/group initially selected for F2 generations to be pruned to 30/sex/group at mating period
Control animals:
yes, sham-exposed
Parental animals: Observations and examinations:
All animals were examined twice daily for viability and overt signs of toxicity. A detailed clinical examination was conducted weekly. body weights were recorded weekly until confirmation of mating. Females were weighed on GD 0, 7, 14 and 21 and LD 0, 7, 14 and 21. Food consumption was measured weekly except during mating, gestation and lactation.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
Litters were examined as soon as possible after delivery for litter size, stillborns, live births and any gross anomalies. Litters were culled by random selection to 8 pups on lactation day 4. culled pups as well as any which died spontaneously during the lactation period were necropsied and examined for anomalies. Pups were weighed individually on LD 0, 4, 7 and 14. On LD21, all pups were counted, sexed and weighed individually with the weights recorded by sex.
Postmortem examinations (parental animals):
At sacrifice, the following organs and tissues were collected for microscopic examination: epididymis, lung, ovary, pituitary, prostate, seminal vesicle, testis, uterus, vagina and regional lymph nodes. Organs were examined in the control and high dose groups only. All masses and gross lesions from all groups were examined. Epididymis, lung, ovary, testis, prostate/seminal vesicle and uterus/vagina were weighed.
Postmortem examinations (offspring):
No data
Statistics:
Fertility indices and male/female sex ratios were compared by the Chi-square test criterion. Proportions of litters with malformations were compared by the Fisher's exact probability test to determine the significance of difference. Proportions of resorbed and dead fetuses, pre- and 10post-implantation losses and pup survival indices were compared by the Mann-Whitney U-test to determine the significance of difference.
Numbers of corpora lutea, total implantations and live fetuses, mean fetal body weights, parental body weights, maternal body weight changes, organ weights and hematological parameters were compared by one-way ANOVA, Bartlett's test for homogeneity of variance and the appropriate t-test for equal and unequal variance using Dunnett's multiple comparison tables to determine the level of significance.

Mean numbers of liveborn pups per litter and mean pup weight were also compared by ANOVA and the appropriate t-test. All statistical analyses compared the treatment groups to control with the levels of significance at p < 0.05 and p < 0.01. All means were accompanied by standard deviations.
Clinical signs:
effects observed, treatment-related
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 specified
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
F0 generation
All F0 males survived to the scheduled sacrifice with minimal clinical evidence of toxicity. Body weight gain was significantly reduced (5-7% in 500 ppm group and 14-16% in 1500 ppm group). 7 females died in the 1500 ppm group (3 prior to mating, 3 during gestation and 1 during lactation). Weight gain was significantly suppressed in the 500 ppm (5-7%) and 1500 ppm (5-7%) groups. Food consumption was reduced in the 1500 ppm group but only in the first week of exposure and was not signficantly different from control animals throughout the rest of the exposure period.
No significant pathological findings in the reproductive organs of F0 animals were observed.
There were no significant differences in fertility indices: number of mated females, number of females delivering a litter, number of females delivering a live litter or male fertility. Mean number of live births/litter was not significantly reduced neither was the length of time for mating.

F1 generation
Birth of offspring from treated dams was not significantly reduced in comparison to controls nor were mean body weights significantly different at LD 4.

F2 generation
Mean body weight changes were similar in the 500 and 1500 ppm group as noted for the F0 generation. Clinical signs of toxicity were noted in the 1500 ppm group (ataxia, reduced motor activity). 6 females in the 1500 ppm group died compared to 1 each in the control group, 100 and 500 ppm groups. Mean number of live offspring/litter and number of live births/number delivered were significantly reduced in the high exposure groups. Mean birth weight of offspring in the high exposure group was also reduced but was not significantly different from control. Male fertility was significantly reduced in the 1500 ppm group but was not considered treatment-related since no such reduction was noted in the F0 and F2 generations.
Dose descriptor:
NOAEC
Effect level:
>= 1 500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: No effect on reproductive indices
Remarks on result:
other: Generation: P, F1 and F2 (migrated information)
Dose descriptor:
NOAEC
Effect level:
ca. 500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Reduced body weights
Remarks on result:
other: Generation: P and F1 (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
F1 generation
Mean body weight was significantly reduced in pups from the 1500 ppm group from LD 7 through the remainder of the weaning period. No diferences in survival indices were noted during the lactation period.

F2 generation
In the F2 offspring, there were few treatment-related effects. Litter size and birth weight were not different from control in the 100 and 500 ppm groups. Mean body weights were not significantly different from control in any group at LD0 or LD4. However weights in the 1500 ppm groups were singificantly lower from LD7 and through the rest of the weaning period. Fraction of liveborn offspring was slightly but significantly reduced in the 1500 ppm group but survival through the rest of weaning was not different from control.
Dose descriptor:
LOAEC
Generation:
F2
Effect level:
ca. 100 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: reduced body weight and survival
Reproductive effects observed:
not specified
Conclusions:
There were no selective reproductive and developmental effects following exposure to C9 aromatic naphtha
Executive summary:

Results from the three-generation reproduction inhalation study in rats indicated limited effects from C9 aromatic naphtha (a C9 aromatic solvent) on the reproductive system up to 1500 ppm. F0 rats were exposed for 10 weeks prior to and two weeks during mating to target exposure levels of 0, 100, 500, or 1500 ppm (actual levels were 0, 103, 495, or 1480 ppm, equivalent to 0, 505, 2430, or 7265 mg/m3) 6 hr/day, 5 days/wk. Females were additionally exposed on gestation days 0-15, and lactation days 5-21. Exposures continued through breeding of the F1 generation. Effects on Parental generations: Parental generations exhibited decreased survival and body weights (F1 & F2). The F1 but NOT F0 or F2 males at the high exposure level showed decreased fertility that may not be related to exposure. No effects were reported on sperm morphology, gestational period, number of implantation sites, or post-implantation loss in any generation. The F2 generation exhibited a LOAEC of 103 ppm (505 mg/m3) based on reduced body weight and survival, while F0 and F1 generations had NOAECs of 495 ppm (2430 mg/m3) for these effects. Effects on Pups: A decrease was observed in F1 mid-dose body weights and F1 and F2 survival (F2: too few to evaluate for body weight due to poor survival in dams). No adverse developmental effects were reported in any generation at any exposure level. The reproductive NOAEC was 1480 ppm (7265 mg/m3), highest concentration tested.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1980
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
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 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
Vehicle:
unchanged (no vehicle)
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.
Dose descriptor:
NOAEC
Effect level:
>= 1 720 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: 300 ppm; No effects to the reproductive system were observed
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.
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
>= 1 720 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: 300 ppm; No effects to the reproductive system were observed
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.

Additional information

READ ACROSS DATA: C14-C20 aliphatics (<2% aromatic)

A reproductive/pre-natal developmental one-generational study was conducted to evaluate the reproductive performance (gonadal function, all 4 stages of the estrus cycle, fertilization, implantation of the egg, gestation period and parturition) of a C16-C30 highly purified light mineral oil (60% paraffins, 40% naphthenes). The pre-natal developmental phase also evaluated pup growth and development up to weaning. Female Sprague-Dawley rats were dermally exposed to test material for 10 weeks premating period (5 days/week), a 3-week mating period (5 days/week) and up to gestation day 20. Test material was applied once daily to clipped, intact dorsal skin at a dose level of 125, 500, 2000 mg/kg bw/day. No clinical signs of systemic toxicity were observed and there were no effects on maternal body weight and food consumption. No differences were observed in mean numbers of implantation sites, live pups/litter, live birth index (total pups born alive/total pups born) either at birth, or on days 4 or 21 postpartum. There was no change in mean pup body weights and no pathological observations were seen in necropsied organs of the pups. NOAEL for mineral oil was ≥ 2000 mg/kg bw/day for fertility and pre-natal development. In males exposed to the same test substance for 13 weeks prior to mating, the reproductive performance was evaluated with regard to fertility, sperm count, sperm morphology and spermatogenesis. No changes in male reproductive organ weights or performance were observed. NOAEL for fertility was ≥ 2000 mg/kg bw/day.

READ ACROSS DATA: C13-C22 aliphatics (<2% aromatic)

F0 and F1 male and female rats were administered an oral gavage dose (50, 200 or 750 mg/kg bw/day) of a C13-C22 hydrocarbon fuel comprised primarily of aliphatic constituents (30% n- and 70% iso-paraffins) 5 days/week for at least 70 days premating and 14 days mating (for males). Test substance had no effect on reproductive performance (fertility), gestation length and parturition in both F0 and F1 rats. There were no test-related effects on postnatal survival, mortality, systemic toxicity, anogenital distance and pup weights in F1 and F2 litters. , NOAEL for fertility was greater than 750 mg/kg bw for males and females.

SUPPORTING DATA: JP-8 Fuel (C9-C16 Aliphatics, 25% Aromatics) 

There were several studies located for the structurally analogous test material, JP-8 fuel. JP-8 fuel was examined for reproductive toxicity in a 70 day male and in a 90 day female one generation reproductive toxicity study (OECD TG 414). For the male reproductive toxicity study, the reproductive NOAEL 3000 mg/kg/day for male rats, which was the highest dose tested. For the female reproductive toxicity study, the reproductive NOAEL 1500 mg/kg/day for female rats, which was the highest dose tested. The F1 (fetus) NOAEL = 750 mg/kg/day based on a decrease in body weight that correlated to a decrease in maternal body weight.

READ ACROSS DATA: C9-14 aliphatics (<2% aromatic) 

 

C9-C14 aliphatic, <2% aromatic hydrocarbon fluids (decane) were examined for reproductive toxicity in a 28-day combined repeated dose toxicity study with the reproduction / developmental toxicity screening test (OECD TG 422).  C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids were administered oral gavage at a dose of 0, 25, 150, or 1000 mg/kg/day to groups of Sprague-Dawley rats. It was concluded that C9-C14 aliphatic, <2% aromatic hydrocarbon fluids did not induce reproductive toxicity in the parental animals and no effects on the endocrine system were observed.  Therefore, the NOAEL was determined to be ≥1000 mg/kg bw/day. 

 

C9-C14 aliphatic, <2% aromatic hydrocarbon fluids (undecane) were examined in a reproduction / developmental toxicity screening test (OECD TG 422).  C9-C14 aliphatic, <2% aromatic hydrocarbon fluids were administered by oral gavage at a dose of 0 (vehicle), 100, 300, 1000 mg/kg/day to groups of Sprague-Dawley rats. It was concluded that C9-C14 aliphatic, <2% aromatic hydrocarbon fluids did not induce reproductive toxicity in the parental animals and no effects on the endocrine system were observed.  Therefore, the NOAEL was determined to be ≥1000 mg/kg bw/day. 

 

READ ACROSS DATA: C9-14 aliphatics (2-25% aromatic) 

 

C9-14 aliphatics (2-25% aromatic) hydrocarbon fluids were examined for reproductive toxicity in a reproduction / developmental toxicity screening test (OECD TG 421). C9-14 aliphatics (2-25% aromatic) hydrocarbon fluids were administered by inhalation at a dose of 0,100, and 300 ppm to groups of rats. It was concluded that C9-14 aliphatics (2-25% aromatic) hydrocarbon fluids did not induce reproductive toxicity in the offspring or in the parental animals. Therefore, the NOAEL was determined to be ≥300 ppm (1720 mg/m3).

Effects on developmental toxicity

Description of key information

One developmental study was available on C16-C20 n-alkanes, isoalkanes, cyclics, <2% aromatics showing a NOAEL>1000 mg/kg/day for both maternal and developmental toxicity.

READ ACROSS DATA: C16-C30 highly purified light mineral oil (60% paraffins, 40% naphthenes)

Pre-natal development toxicity (Similar to OECD TG 414) - Dermal, oral and inhalation exposure (gestation days 6-19). NOAEL for developmental toxicity was 2000 mg/kg bw/day (dermal), 1000 mg/m3 (inhalation) and 5000 mg/kg bw/day (oral gavage).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 29, 1995 to June 29, 1995.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Some deviations such as the treatment period (from GD6 to GD15 instead of GD5 to GD15 mentioned in OECD guideline 414).
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
females were dosed from Gestation Day (GD) 6 instead of GD 5; Administration volume 5 mL/kg (instead of 4mL/kg max).
Principles of method if other than guideline:
Guideline study
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston Facility, New York.
- Age at study initiation: females were approximately 10 weeks at GD 0
- Weight at study initiation: 216 to 295 g at GD 0
- Housing: Single housed during the study period, except during mating
- Diet: Certified Rodent Diet (5002 meal), ad libitum
- Water: ad libitum (Elizabethtown Water company)
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24.4°C
- Humidity (%): 40 - 70%
- Air changes (per hr): Not mentioned
- Photoperiod: 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): OCT1295B
- Supplier: Best Foods, CPC International
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At analytical chemistry analysis, excellent uniformity was observed. The percent relative standard deviation (%RSD) ranged from 1.43% to 2.97%. Stability data indicated that the test material was stable in corn oil at ambient temperature for at least 13 days. Concentration verification analysis indicated that the solution in corn oil was within 12% of the nominal concentrations for Week 1 and Week 3.
Details on mating procedure:

- M/F ratio per cage: 1:1 (male:female) ratio
- Length of cohabitation: one night
- Proof of pregnancy: Mating was confirmed by observation of a copulatory plug (vaginal) and/or by the presence of sperm in a vaginal rinse. The day on which mating was confirmed was the female's Day 0 of gestation (GD 0).
Duration of treatment / exposure:
from Gestation Day (GD) 6 through GD 15
Frequency of treatment:
Once daily
Duration of test:
Inlife test period: May 29, 1995 to June 29 1995.
Remarks:
Doses / Concentrations:
0, 400, 800 and 1000 mg/kg/day
Basis:
other: ingested doses
No. of animals per sex per dose:
25 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on a rangefinding study with Exxsol D130. Results of this study indicated no overt or consistent signs of toxicity at 1000 mg/kg (the limit dose for developmental studies) (OECD, 1981).
- Rationale for animal assignment (if not random): sequential Physical Identification Number
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily during the treatment period, otherwise at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior the selection and daily during gestation

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, 6, 9, 12, 15, 18, and 21

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: All females assigned to groups were examined by gross necropsies.

OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: [half per litter ]
Statistics:
Statistical treatment of the results was conducted where appropriate. Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test for ordered response in the dose groups. First, Bartlett's Test was performed to determine if the dose groups had equal variance . If the variances were equal, the testing was done using parametric methods (ANOVA + Dunnett's), otherwise nonparametric techniques were used (Kruskl-Wallis + Dunn's Summed Rank Test).
Fetal malformation and variation incidence data were analyzed for statistical significance as follows: First, a standard chi-square analysis was performed to determine if the proportions of incidences differ between the groups tested. Next, each treatment group was compared to the control group using a 2 x 2 Fisher Exact Test.
Indices:
Maternal data:
Percentage of pre-implantation loss
Percentage of post-implantation loss
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No maternal toxic effects.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were statistically significant differences in individual skeletal variations, but were limited to an increase in the incidence of rudimentary lumbar ribs in the 400 mg/kg dose group (14.2%) compared with controls (4.2%) on a per fetus basis. This increased incidence was within the historical control range of this laboratory (3.7-28.8), greater than that observed at higher doses, and thus, not considered biologically important.
The statistically significant increases in the total fetuses with skeletal variations in the 400 mg/kg dose groups were directly driven by the increased incidence of rib variations and thus, were similarly considered as biologically unimportant (within HC range).
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Abnormalities:
no effects observed
Developmental effects observed:
no

No remarks

Conclusions:
In conclusion, overt signs of maternal toxicity were not apparent at any dose tested, as indicated by the absence of adverse clinical or postmortem findings or effects on body weight, food consumption, and/or uterine implantation data. Similarly, there were no treatment-related adverse effects on fetal development or growth observed at any dose level tested. Accordingly, the maternal and developmental NOAELs (No Observable Adverse Effect Level) were established at 1000 mg/kg (the limit dose) under the conditions of this study.
Executive summary:

The test material was administered by oral gavage to three groups of Crl:CDBR female rats at doses of 400, 800, and 1000 mg/kg/day. A fourth group (Group 1) served as a control and received the carrier (corn oil) only. Mated females were dosed once daily from Gestation Day (GD) 6 through GD 15. Dosing volumes were 5 mL/kg for all groups and based on the animals' most recent body weights.

There was no evidence of overt systemic maternal toxicity at any dose level tested. overt signs of maternal toxicity were not apparent at any dose tested, as indicated by the absence of adverse clinical or postmortem findings or effects on body weight, food consumption, and/or uterine implantation data.

Similarly, there were no treatment-related adverse effects on fetal development or growth observed at any dose level tested. There was a statistically significant increase in the incidence of rudimentary ribs in the low dose (400 mg/kg/day) group, but not at the higher doses, compared with controls, which resulted in an increased incidence of total fetuses with skeletal variations in the 400 mg/kg/day group. However, all these incidences were within the historical control range of this laboratory. Therefore, this common finding in fetal rats was not considered biologically important.

Accordingly, the maternal and developmental NOAELs (No Observable Adverse Effect Level) were established at 1000 mg/kg/day under the conditions of this study.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report equivalent or similar to OECD guideline 414:
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Pregnant Sprague-Dawley rats by single dose exposures through three separate routes of exposure; inhalation (1000 mg/m3), dermal (2000 mg/kg bw/day), oral gavage (5000 mg/kg bw/day) with exposures on gestation days 6-19.
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Lakeview, NJ
- Diet: Purina Certified Rodent Chow #5002 (Meal)
- Water: Tap water via automatic water system ad libitum
- Acclimation period: 3 weeks
- Age at study start - Approximately 12 weeks
Route of administration:
other: Oral gavage, dermal and aerosol inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
water
Details on exposure:
Dermal application
Stock 461 was applied once daily to the clipped, intacti dorsal skin of the rat at a dose level of 2000 mg/kg body weight/day (maximum practical dose dermally applicable). Application sites were not covered. Stock 461 was drawn up into a 1 cc syringe (calibrated in 0.01 cc) and dispensed evenly on the the clipped skin using the tip of the syringe without the needle. To minimize ingestion, rats were fitted with cardboard Elizabethan-style collars on gestation day 0 and replaced as necessary. Collars were lined with latex tubing to minimize the development of irritation or lesions. Rats were clipped on gestation day 5 and again on day 12.

Inhalation
Presumed-pregnant rats were exposed to 1 mg/l (1000 mg/m3) of stock 461 for 6 hr/day on days 6-19 of gestation. Aerosol was generated by stainless steel Laskin nebulizers inside a glass vessel containing test substance. Setup was designed to allow the maximum proportion of respirable particles (< 5 um in diameter) to pass into the exposure chamber while larger particles impacted the walls of the glass vessel and deposited. Airstream was passed through an elutriator to remove majority of remaining large aerosol droplets. Aerosol-laden air was then mixed with incoming airstream before entering the exposure chamber. Exposures were performed in 400-liter chambers with individually housed animals. Airflow was set for at least 12 air changes per hour.

Oral gavage
21 presumed-pregnant rats were orally dosed with 5000 mg/kg body weight/day of test substance on days 6-19 of gestation. Rats were dosed with a 3 cc syringe fitted with a 16-gauge stainless steel gavage needle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Oral and dermal applications - Amount of stock administered to each rat was calculated using the most recently recorded body weight for each animal, dose level and density of test substance

Inhalation exposure - Mean aerosol concentration determined gravimetrically matched the target concentration of 1 mg/l. Mass median aerodynamic diameter of particles was 1.2 um with a geometric standard deviation of 1.8 um. Particles were determined to be weill within respirable range (less than 5-10 um)
Details on mating procedure:
During the mating period, female rats which had not previously borne pups were placed with adult male rats from their corresponding treatment group in a ratio of 1:1 and observed daily for evidence of having engaged in breeding activity. Each morning during the period of cohabitation, the drop-pan papers under the animal cages were checked for the presence of expelled vaginal sperm plugs; additionally, each female rat was examined for the presence of in situ vaginal sperm plugs. Vaginal lavage fluid was obtained from each female which exhibited a vaginal sperm plug in situ or on the drop-pan papers, and was examined for the presence of spermatozoa. Females that were positive for sperm plug as well as for spermatozoa were considered to be at day 0 of presumed gestation and were placed in individual housing units. The cohabitation was continued until 122 presumed-pregnant female rats were obtained.
Duration of treatment / exposure:
Gestation days 6-19
Frequency of treatment:
Inhalation - 6 hr/day

Oral/dermal - Once/day
Duration of test:
Inlife test period - Feb 12-23 1985 to March 5-15, 1985
Remarks:
Doses / Concentrations:
2000 mg/kg bw/day
Basis:
other: Dose dermally applied
Remarks:
Doses / Concentrations:
5000 mg/kg bw/day
Basis:
actual ingested
Oral
Remarks:
Doses / Concentrations:
1000 mg/m3
Basis:
nominal conc.
Inhalation exposure
No. of animals per sex per dose:
Dermal application - 20 rats/group
Oral gavage - 21 rats/group
Inhalation exposure - 20 rats/group
Control animals:
yes, sham-exposed
Maternal examinations:
All animals in each group were monitored at least once a day throughout gestation until sacrifice for changes in appearance, behavior, excretory function, ill-health, mortality or abortion. Body weight of each presumed-pregnant female was measured to the nearest 0.1 gram on days 0, 6, 8, 10, 13, 16, 18 and 20 of gestation. Food consumption was measured for gestation day intervals of 0-6, 6-8, 8-10, 10-13, 13-16, 16-18 and 18-20.
Ovaries and uterine content:
Ovaries and uterus of each rat were excised and examined grossly. Number of corpora lutea per ovary were counted and recorded. Uterine content of each pregnant rat were exposed and the number and location of implantations, early, mid and late resorptions, live and dead fetuses were recorded.
Fetal examinations:
Each live fetus was gendered, weighed and measured for crown-rump distance and grossly examined for external anomalies. Fetuses in each litter were distributed randomly into 2 groups: one half fixed in Bouin's solution and examined for soft tissue anomalies. The other half were peeled, eviscerated, fixed in 95% ethanol, macerated in pottasium hydroxide, differentially stained for cartilage and bone, cleared in glycerin and examined for skeletal anomalies.
Statistics:
Maternal biophase and cesarean section data and fetal data were evaluated by ANOVA followed by group comparisons using Fisher's exact or Dunnett's test. Differences following comparison of experimental data were considered statistically significant at p value < 0.05.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Dermal irritation (erythema, flaking and scabbing) of skin was noted in the dermally exposed rats. MAternal parameters such as food consumption and body weight gain were not adversely affected by test substance exposure. Reproductive parameters such as number of implants, number of resorptions, number of viable fetuses were not affected by test substance exposure.
Dose descriptor:
NOAEL
Effect level:
>= 2 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 5 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
>= 1 000 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal parameters such as body weight, crown-rump length were changed compared to controls. There was no evidence of teratogenicity in external, skeletal and visceral evaluations of fetuses.
Dose descriptor:
NOAEL
Effect level:
>= 2 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
>= 5 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEC
Effect level:
>= 1 000 mg/m³ air (nominal)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
NOAEL for teratogenicity was ≥ 2000 mg/kg bw/day (dermal), 1000 mg/m3 (inhalation) and 5000 mg/kg bw/day (oral gavage).
Executive summary:

A series of studies were conducted to evaluate the teratogenic effects of a C16-C30 light mineral oil in pregnant Sprague-Dawley rats by three separate routes of exposure; inhalation (1000 mg/m3), dermal (2000 mg/kg bw/day), oral gavage (5000 mg/kg bw/day) with exposures on gestation days 6-19. Due to low vapor pressure, the mineral oil was delivered as an aerosol for the inhalation study though aerosol particle size generated was well within the respirable range. There were no observable maternal effects (food consumption, body weight gain) irrespective of route of exposure. No adverse effects on reproductive parameters (implantation number, resorptions, fetal viability) or fetal parameters (body weight, crown-rump length) were observed in any of the studies. There was a clear lack of teratogenicity with regard to abnormal ossification, and adverse effects to external or soft tissue. Spontaneous anomalies were noted in some exposure groups but were not considered exposure related since incidence of such occurrences were similar in the sham-exposed groups. NOAEL for developmental toxicity was ≥ 2000 mg/kg bw/day (dermal), 1000 mg/m3 (inhalation) and 5000 mg/kg bw/day (oral gavage).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
3 September 2010 - 20 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report equivalent or similar to OECD guideline 414: GLP
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Source - Charles River (UK) Ltd
Age at arrival - 65 days
Weight range - 227 - 254 g
Age at study start - 70 days
Weight range at study start - 224 - 272g

Temperature - 19-23 deg C
Relative humidity - 40 - 70%
Light cycle - 12/12 hrs light/dark

Diet and water provided ad libitum
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Animals received the test material or vehicle control formulations orally at a volume-dose of 4 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of the test material in the liquid matrix was demonstrated as 15 days at 2-8°C (refrigerated) and 24 hours when stored at ambient temperature in Huntingdon Life Sciences Study Number: HJL0145.
During the first and last weeks of treatment, the test formulations were analysed for achieved concentration of the test substance. Four samples (nominally 1 mL accurately weighed) were taken from all groups. Two assays from each group were analysed and the remainder were retained refrigerated (nominally 2-8 ºC) as contingency.
Details on mating procedure:
Animals were paired 1:1 male/female ratio. Daily checks were made after pairing for evidence of mating, including ejected copulation plugs in cage trays and the presence of sperm in a vaginal smear.
Animals were allocated to study on Day 0 of gestation, when positive evidence of mating was detected. Only females with a sperm positive vaginal smear or at least two copulation plugs were selected.
Duration of treatment / exposure:
Gestation days 6-19
Frequency of treatment:
Once daily
Duration of test:
Gestation day 0-20
Remarks:
Doses / Concentrations:
0, 100, 300, 1000
Basis:
actual ingested
No. of animals per sex per dose:
20/dose
Control animals:
yes, concurrent vehicle
Maternal examinations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health. The weight of each adult female was recorded on Days 0, 3 and 6-20 after mating. The weight of food supplied to each adult female, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.
Ovaries and uterine content:
The gravid uterus was weighed before removal of the fetuses; this weight included the weight of the cervix, uterus, fallopian tubes and ovaries. For each animal, the number of corpora lutea in each ovary and the number of implantation sites, the number and distribution of resorption sites (classified as early or late) and live and dead fetuses were recorded for each uterine horn.
Fetal examinations:
All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each fetus and placenta was externally examined and any abnormalities were recorded. The sex of each fetus was recorded. Approximately half of the fetuses in each litter were eviscerated and their skeletons were fixed in Industrial Methylated Spirit, prior to processing and staining with Alizarin Red and Alcian Blue. The remaining fetuses were fixed whole in Bouin’s fluid. Free-hand serial sections were prepared from the Bouin’s fixed fetuses and were examined under the microscope for visceral abnormalities. Fetuses stained with Alizarin Red and Alcian Blue were assessed for skeletal and cartilage development and abnormalities.
Statistics:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose-response was not monotone, Dunnett's test was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

For gravid uterine weight and litter data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable. Pre/post implantation loss and sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect. Each treated group was compared to control using a Wald chi-square test. For resorptions, each treated group was compared to control by exact rank Wilcoxon rank sum test.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No mortalities were recorded in study. Body weight and bodyweight gain was not affected by treatment with test substance. Mean body weight gain was statistically significantly lower compared to controls on days 14-15 and 15-16 but were not considered of biological relevance. Relative gravid uterine weight was not changed and no macropathological findings related to treatment were observed.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no effect of treatment on mean numbers of implantations, embryo-fetal resorptions or live fetuses, or sex ratio or pre- and post-implantation loss. Mean fetal, placental or litter weights were unchanged compared to controls and incidence of major and minor abnormalities and skeletal variations showed no relationship to treatment.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Oral administration of MCP 2484, (a base oil), to Crl:CD(SD) rats during the organogenesis phase of embryo fetal development resulted no adverse effects being observed at dose levels upto 1000 mg/kg/day.
It was concluded from this study that the dosage of 1000 mg/kg/day was the maternal no observed-adverse-effect-level (NOAEL) and 1000 mg/kg/day was the no-observed-adverseeffect-level (NOAEL) for embryo fetal survival and development.
Executive summary:

A pre-natal developmental toxicity study was conducted to evaluate the fetal developmental effects of a C24 alkylated naphthalene administered by oral gavage. No developmental effects were observed and NOAEL was ≥ 1000 mg/kg bw/day, the highest dose tested.

Endpoint:
developmental toxicity
Type of information:
experimental study
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:
read-across: supporting information
Qualifier:
equivalent or similar to
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 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.
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.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: fetotoxicity
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.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May 3, 1991 - September 18, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report equivalent or similar to OECD guideline 414
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Exposure from GD 6-15 rather than 20 with rats sacrificed on day 21
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Source - Not stated
Purchase order/Shipping reference number - 1GM34339/39119598
Age at animal reciept - 10 weeks (males), 11 weeks (females)
Weight at initiation of mating - Approximately 242 - 340 grams (at designated day 0)
Acclimation period - 21 days

Housing - Individual except during mating
Feed (ad libitum) - Purina certified rodent chow (502 meal) - Ralston Purina Company, Checkerboard square, St. Louis, MO, 63164
Water - ad libitum
Temperature - 68-76 deg F
Humidity - 40 - 70% relative humidity
Lighting - 12/12 hours light/dark cycle
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Neat test material was diluted in carrier and thoroughly mixed prior to dispensing. Mated females were dosed once daily by oral gavage from GD 6 through GD 15. Dose levels were based on the most recent body weights. Dosing volumes were 5 ml/kg for all groups. Animals were dosed approximately the same time each day (plus or minus one hour).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose solution mixtures of MRD-90-884 in corn oil demonstrated uniformity. Concentration verification analyses indicated that sample concentrations were within 10% of the target values at all dose levels. Stability was demonstrated on the 1.5% nominal concentration levels for up to 8 days at
room temperature.
Details on mating procedure:
Males and females were paired in a 1:1 ratio in cages. Mating was confirmed on the following morning by observation of a copulatory plug in the female's vagina or by the presence of sperm in a vaginal rinse. The day on which mating was confirmed was the female's Day 0 of gestation (GD 0). After confirmation of mating, each mated female was returned to its own cage. Mated females were assigned to dose groups in order of mating.
Duration of treatment / exposure:
Gestation day 6-15
Frequency of treatment:
Once daily gavage
Duration of test:
Gestation day 0 - 21
Remarks:
Doses / Concentrations:
0, 75, 150, 450 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25/group
Control animals:
yes, concurrent vehicle
Maternal examinations:
The animals were examined,for viability twice daily during the treatment period and at least once daily at other times during the study. Body weights were recorded prior to selection, weekly during the mating period, and for each mated female assigned to a study group, on GD 0, 6, 9, 12, 15, 18, and 21G. Food consumption was measured concurrently with body weights during gestation. A clinical examination was given to each female prior to selection and daily thereafter.
Ovaries and uterine content:
Uterine weight with ovaries attached was measured at necropsy. Uterine contents were examined and the numbers and locations of implantation sites, early and late resorptions, live, dead, and externally malformed fetuses were recorded. Corpora lutea were counted and recorded. The uteri of all apparently non-pregnant dams were stained to confirm pregnancy status.
Fetal examinations:
Each live fetus was weighed and examined externally for gross malformations, including cleft palate. All live fetuses were euthanized by intramuscular injection into the tongue with sodium pentobarbitol prior to internal examination. The sex of each live fetus was determined by external examination and confirmed internally only on those fetuses receiving visceral examinations. Approximately one-half of the fetuses of each litter were decapitated. These heads were preserved in Bouin's solution for at least two weeks. Free-hand razor blade sections of the Bouin's-fixed fetal heads were examined for the presence of abnormalities. The viscera of all decapitated fetuses also were examined by fresh dissection. The remaining fetuses were eviscerated, processed for skeletal staining with Alizarin red, and examined for the presence of malformations and ossification variations.
Statistics:
Bartlett's test of homogeneity of variance was used to determine if the groups had equivalent variances at the 1% level of significance.

1. If the variances were equivalent, the hypothesis that there was no difference in response between the groups was tested using a standard one-way analysis of variance. If the ANOVA was significant, Dunnett's test was performed to determine which treated groups differed from the control. A
linear regression to test for a dose response was also performed and tested for lack of fit to the regression.

2. If the variances were not equivalent, then a Kruskal-Wallis (non-parametric) test was performed to determine if the treatment effects were equivalent. If there was a difference, a rank sum comparison was used to determine which treatment groups differed from the control. Jonckheere's test for ordered response also was performed. The following were calculated and analyzed for statistical significance as described below:

incidences of individual and total malformations
incidences of individual and total variations

A standard chi-square analysis was performed to determine if the proportion of incidences differed between the groups tested. Each treatment group was then compared to each control group using a 2 x 2 Fisher Exact test. Armitage's test for linear trend in the dosage groups was performed.
Indices:
mean body weight and gravid uterine weight of pregnant animals
mean body weight change of pregnant animals
mean food consumption of pregnant animals
mean number of corpora lutea per dam
mean number of implantations per dam
mean number of resorptions per dam
mean number of live fetuses per litter
mean number of live male fetuses per litter
mean number of live female fetuses per litter
mean number of dead fetuses per litter
mean number of malformed fetuses per litter
mean number of affected (resorbed, dead or malformed) offspring per litter
mean number of fetuses with developmental variations per litter
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Decreased mean body weight gain and statistically significant decrease in mean maternal food consuption on gestation days 6-9.
Dose descriptor:
NOAEL
Effect level:
ca. 150 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean fetal body weights for treated groups were coomparable to controls. No meaningful differences in the incidences of fetal variations or malformations were observed in treated groups compared to controls. Mean number of ossification sites in treated groups were comparable with controls.
Dose descriptor:
NOAEL
Effect level:
>= 450 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
C10-C12 aromatic hydrocarbon solvent is not a developmental toxicant
Executive summary:

In a developmental study, pregnant dams were dosed by oral gavage with 75, 150 or 450 mg/kg/day of a C10-C12 aromatic hydrocarbon solvent during gestational days 6 through 15. At 450 mg/kg/day, maternal body weight gain and food consumption were significantly decreased during the first three days of treatment. No adverse fetal effects were noted at any dose level. Thus, C10-C12 Aromatic fluids did not produce any fetal toxicity or any developmental effects in rats. Based on the study results, the maternal NOAEL is 150 mg/kg/day and the developmental NOAEL is greater than 450 mg/kg/day

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Accepted, well conducted study. Exposure period from GD 6-15
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Exposure on GD 6-15
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
Source - Charles River Labs, Portage, MI
Age at receipt - 8 weeks
Acclimation period - 2 or 3 weeks
Feed - Purina certified Rodent chow #5002 (available except during exposure)
Water - Ad libitum
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
All animals were exposed in 16 cubic meter glass and stainless steel chambers. Chamber ventilation was provided by an HVAC system separate from the general laboratory air handling system. This air was particulate-filtered and controlled for temperature and humidity. Chamber air flow rate, temperature, and relative humidity were monitored every half-hour during exposure periods.

To generate test atmospheres, nitrogen was heated to 200 deg C by passage through a 1-liter stainless steel cylinder fitted with a 1500-watt band heater and was then introduced at the bottom of a glass column 7.6 cm in diameter and 30 cm long, packed with glass beads. The liquid test material was delivered by a fluid metering pump from a stainless steel safety can, through Teflon tubing, to the bottom quarter of the column. The test sample was vaporized as it flowed up the column with the nitrogen. The vapors were passed to the chamber inlet where dilution with chamber ventilation air reduced the concentration to the desired exposure levels.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure levels were monitored with a gas-phase infrared spectrometer. Measurements were made on an hourly basis throughout the study. Accuracy of this method was confirmed by the use of vapor standards. Additionalyy, the composition (on a weight percentage basis) of the test material within each exposure chamber was determined by gas chromatography during the first exposure week.
Details on mating procedure:
Female mice were paired 1:1 with male mice for mating. Female mice were confirmed to have been mated by visual presence of a vaginal plug.
Duration of treatment / exposure:
Gestation day 6-15
Frequency of treatment:
6 hrs/day
Duration of test:
Gestation day 0-18
Remarks:
Doses / Concentrations:
102 +/- 2.6 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
500 +/- 3.7 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1514 +/- 22.9 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
30 mice/group
Control animals:
yes, sham-exposed
Details on study design:
Mated female mice were randomly assigned to one of four treatment groups, each containing 30 animals. Contrl group was exposed to room air only.
Maternal examinations:
Mice were examined twice daily for viability and overt changes in appearance and behavior. Presence and duration of clinical signs were recorded daily from GD6-GD15 and also on GD18. Maternal body weight was measured on GD0 and daily from GD6 to GD18.
Ovaries and uterine content:
Surviving females were sacrificed on GD18. Uterine contents were examined for the number and location of viable and non-viable fetuses, early and late resorptions. The number of total implantations and corpora lutea were recorded. Uteri were excised and weighed.
Fetal examinations:
Fetuses were individually weighed, sexed, tagged and examined for external malformations and variations. Approximately half of the fetuses were dissected, internally sexed and examined for visceral malformations and variations.
Hearts were dissected by a modification of the method described by Staples (1974). Heads were fixed in Bouin's solution for soft tissue examination by the razor blade sectioning technique. Remaining fetuses were fixed in alcohol, macerated with potassium hydroxide, stained with Alizarin Red S and cleared with glycerin. Features were subjected to skeletal examination.
Statistics:
Fertility indices and male/female sex ratios were compared by the Chi-square test criterion. Proportions of litters with malformations were compared by the Fisher's exact probability test to determine the significance of difference. Proportions of resorbed and dead fetuses, pre- and post-implantation losses and pup survival indices were compared by the Mann-Whitney U-test to determine the significance of difference.
Numbers of corpora lutea, total implantations and live fetuses, mean fetal body weights, parental body weights, maternal body weight changes, organ weights and hematological parameters were compared by one-way ANOVA, Bartlett's test for homogeneity of variance and the appropriate t-test for equal and unequal variance using Dunnett's multiple comparison tables to determine the level of significance.

Mean numbers of liveborn pups per litter and mean pup weight were also compared by ANOVA and the appropriate t-test. All statistical analyses compared the treatment groups to control with the levels of significance at p < 0.05 and p < 0.01. All means were accompanied by standard deviations.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The highest concentration (1500 ppm; nominal concentration) produced severe maternal toxicity. Mortality in this group was 44% (12 mice died between GD 8-16 and 2 on GD 6). Mean maternal body weight gain in the 1500 ppm group was significantly reduced during exposure periods (GD 6-15) and from GD0-18. clinical observations made in this dose group included abnormal gait, labored breathing, hunched posture, weakness, inadequate grooming, circling and ataxia.

In the 500 ppm group, 2 mice died during the exposure period with one due to treatment-unrelated injury and the other undetermined. Maternal body weight gain in the 500 ppm group showed a statistically significant reduction compared to chamber controls. there were no statistically significant reductions in body weight gain in the 100 ppm group and clinical signs of toxicity were absent in both the 100 and 500 ppm groups.
There were no statistically significant differences in maternal organ weights in any exposure group. Hematological evaluations revealed significant decreases in percent hematocrit and mean corpuscular volume values in the 1500 ppm group compared to controls. Leukocyte count was reduced in the 500 ppm group but was not considered toxicologically relevant since no such effect was noted in the 1500 ppm group.
Dose descriptor:
NOAEC
Effect level:
ca. 100 ppm (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Number of live fetuses per litter and the mean fetal body weight were significantly reduced in the 1500 ppm group. Post-implantation loss was significantly elevated, ossification was delayed particularly in the skull and sternebrae and number of fetuses with cleft palate was substantially increased.
Fetal body weight was significantly reduced in the 500 ppm group with no other evidence for developmental toxicity, malformation or developmental delay. Number of live fetuses/litter was significantly reduced in the 100 ppm group but was considered toxicologically irrelevant as there was no such effect in the 500 ppm group.
Dose descriptor:
NOAEC
Effect level:
ca. 100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
No selective developmental effects were noted. C9 aromatic naphtha only induced developmental effects at maternally toxic exposures.
Executive summary:

Inhalation developmental studies have been conducted on the following category members. In a study with C9 aromatic naphtha (a C9 aromatic solvent), mice were exposed to 0, 100, 500, 1500 ppm from GD 6 -15. In this study, conducted in mice exposed to C9 aromatic naphtha, anomalies (delayed sternebral and cranial ossification) and definitive birth defects (increased incidence of cleft palate) were noted in the presence of severe maternal toxicity (44% mortality) at the highest concentration tested (1500 ppm).  The increased incidence of cleft palate in the pups has been shown to be related to stress in the dams at toxic dose levels (1500 ppm – 44% mortality). Reduction in fetal body weights in the 500 ppm group also occured in the presence of statistically significant reductions in maternal body weight at the same dose level. Overall, exposure resulted in some fetal effects in the presence of maternal toxicity, but no selective developmental effects. The fetal NOAEC values range from 100 to 300 ppm and maternal NOAECs range from 100 to 300 ppm (500 - 1500 mg/m3).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-03-27 to 1989-04-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it followed a protocol comparable to OECD Guideline 414.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: 42 days at arrival
- Weight at study initiation: 30 g male, 24 g females
- Housing: individually in stainless steel wire mesh cages, identified with toe clips and ear notches
- Diet (e.g. ad libitum): Prolab Certified Rodent Food, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66-72 degree F
- Humidity (%): 50-71
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark


IN-LIFE DATES: From: April 5, 1989 To: April 18, 1989
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4320 l glass and stainless steel chambers.
- Method of holding animals in test chamber: cages
- Source and rate of air: 1000 l/min
- Method of conditioning air: Test substance was metered from a piston pump into one or two heated glass evaporator with a temperature of 27-70 degree C. Conditioned air was passed through the evaporator, where it carried the vapor into the exposure chamber.
- Temperature, humidity: monitored every 30 minutes
- Air flow rate: 1000 l/min
- Air change rate: 20 min, 14 air changes per hour
- Treatment of exhaust air: filtration


TEST ATMOSPHERE
- Brief description of analytical method used: GC with flame ionization detection
- Samples taken from breathing zone: yes, 7 times per exposure
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken seven times per exposure period and analyzed with GC-FID. Distribution of test substance was evaluated by sampling five different areas of the exposure chamber.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: March 27, 1989-April 2, 1989
- Proof of pregnancy: vaginal plug referred to as day 0
Duration of treatment / exposure:
gestation day (GD) 6-15
Frequency of treatment:
6 hrs/day
Duration of test:
GD 18
Remarks:
Doses / Concentrations:
0, 900, 3000, 9000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
914, 3026, 9107 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
30 pregnant females per exposure group
Control animals:
yes
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, clinical signs



BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 9, 12, 15


FOOD CONSUMPTION: Yes

WATER CONSUMPTION: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 18
- Organs examined: gravid uterus, ovaries, cervix, vagina, abdominal cavities, thoracic cavities, liver, kidneys


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live and dead fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter were examined for thoracic and abdominal visceral abnormalities
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
Statistics:
Quantitative continuous variables were compared by use of Levene's test for equal variance, analysis of variance, and t-tests with Bonferroni probabilities. Nonparametric data was evaluated using the Kruskal-Wallis test, followed by the Mann-Whitney U test. Indices were compared using Fisher's exact test. 0.05 was used as the criteria for statistical significance.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no significant treatment related effects to body weight, clinical signs, food consumption, weight changes, or organ weights. There was increased water consumption on GD 6-9, 9-12, 6-15, and 15-18 in the 3000 ppm group. There was also increased water consumption in the 900 ppm group on GD 3-6 and 6-9. There was a statistically significant increase in lung color changes in the 9000 ppm group. Four dams also had brown foci. Two dams in the 3000 ppm group had lung color changes as well, and three had dark brown foci.
Dose descriptor:
NOAEC
Effect level:
900 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEC
Effect level:
3 000 ppm
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Gestational parameters were similar between exposure and control groups. There was a statistically significant increase in two skeletal malformations in the 9000 ppm group, bilateral bone island at the first lumbar arch, all intermediate phalanges of the hindlimb unossified. No other dose related abnormalities were noted.
Dose descriptor:
NOAEC
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Dose descriptor:
LOAEC
Effect level:
9 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Results of Developmental Toxicity Study on Mice

0.0 ppm

900.0 ppm

3000.0 ppm

9000.0 ppm

No. of dams with lung color change

0

0

2

12

All inter. Phalanges (hindlimb) unossified (litters, %)

76.9

72.0

84.0

100.0

Bone island - first lumbar arch - bilateral  (litters, %)

0.0

0.0

8.0

23.1

Conclusions:
In mice, the maternal NOAEC was 900 ppm, and the maternal LOAEC was 3000 ppm (10560 mg/m3) based on color changes in the lungs. The developmental NOAEC was 3000 ppm and the LOAEC was 9000 ppm(31680 mg/m3) in mice.
Executive summary:

The purpose of this study was to examine the developmental toxicity of commercial hexane in mice. Groups of 30 pregnant female mice were exposed to concentrations of 0, 900, 3000, or 9000 ppm for 6 hrs/day during gestational days 6 -15. The animals were then sacrificed on GD 18. During the study, the animals were examined for clinical signs, mortality, food and water consumption, and body weights taken. After sacrifice, the internal organs were examined, and the uterus was examined for viable fetuses, number of resorptions, and number of corpora lutea. Fetuses were examined for malformations. Necropsy revealed color changes in the lungs of females in the 3000 and 9000 ppm groups. Fetuses in from dams in the 9000 ppm group had a statistically significant increase in some skeletal abnormalities. The maternal NOAEC in mice was 900 ppm (3168 mg/m3), and the LOAEC 3000 ppm based on lung color changes. The developmental NOAEC in mice was 3000 ppm (10560 mg/m3) and the LOAEC 9000 ppm (31680 mg/m3) based on skeletal abnormalities.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989-03-24 to 1989-04-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it followed a protocol comparable to OECD Guideline 414.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: 63 days males, 56 days females at arrival
- Weight at study initiation: 250-300 g male, 175-200 g females
- Housing: individually in stainless steel wire mesh cages, identified with ear tags
- Diet (e.g. ad libitum): Prolab Certified Rodent Food, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 63-74 degree F
- Humidity (%): 40-71
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark


IN-LIFE DATES: From: April 9, 1989 To: April 21, 1989
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4320 l glass and stainless steel chambers.
- Method of holding animals in test chamber: cages
- Source and rate of air: 1000 l/min
- Method of conditioning air: Test substance was metered from a piston pump into one or two heated glass evaporators with a temperature of 27-70 degree C. Conditioned air was passed through the evaporators, where it carried the vapor into the exposure chamber.
- Temperature, humidity: monitored every 30 minutes
- Air flow rate: 1000 l/min
- Air change rate: 20 min, 14 air changes per hour
- Treatment of exhaust air: filtration


TEST ATMOSPHERE
- Brief description of analytical method used: GC with flame ionization detection
- Samples taken from breathing zone: yes, 7 times per exposure
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken seven times per exposure period and analyzed with GC-FID. Distribution of test substance was evaluated by sampling five different areas of the exposure chamber.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: April 2, 1989-April 6, 1989
- Proof of pregnancy: vaginal plug referred to as day 0
Duration of treatment / exposure:
gestation day (GD) 6-15
Frequency of treatment:
6 hrs/day
Duration of test:
GD 21
Remarks:
Doses / Concentrations:
0, 900, 3000, 9000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
914, 3026, 9107 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
25 pregnant females per exposure group
Control animals:
yes
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 9, 12, 15, 18, 21

FOOD CONSUMPTION: Yes

WATER CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: gravid uterus, ovaries, cervix, vagina, abdominal cavities, thoracic cavities, liver, kidneys


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live and dead fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter were examined for thoracic and abdominal visceral abnormalities
- Skeletal examinations: Yes: half per litter
Statistics:
Quantitative continuous variables were compared by use of Levene's test for equal variance, analysis of variance, and t-tests with Bonferroni probabilities. Nonparametric data was evaluated using the Kruskal-Wallis test, followed by the Mann-Whitney U test. Indices were compared using Fisher's exact test. 0.05 was used as the criteria for statistical significance.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no treatment related effects to mortality or pregnancy rates. In the 9000 ppm exposure group, there was a significant reduction in weight gain on GD 6-9, and GD 6-15, and slightly reduced on GD 9-12. In the 3000 ppm group, there was a significant reduction in weight gain on GD 9-12, but significantly increased on GD 18-21. Food consumption was significantly reduced in the 9000 ppm group on days 6-9, 9-12, 12-15, and 6-15. There were color changes in the lungs of females in the 9000 ppm group. These changes were also seen in one female each in the 0, 900 and 3000 ppm groups. The color changes in the 900 and 3000 ppm groups were not considered treatment related. There were no treatment related effects to gestational parameters.
Dose descriptor:
NOAEC
Effect level:
3 000 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEC
Effect level:
9 000 ppm
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no treatment related effects to the development of fetuses.
Dose descriptor:
NOAEC
Effect level:
9 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Gestational Body Weight Changes (g)

0.0 ppm

900.0 ppm

3000.0 ppm

9000.0 ppm

Day 0-6

 28.21 (11.819)

31.55 (6.463)

31.19 (9.344)

29.36 (8.245)

Day 6-9

11.55 (8.103)

9.12 (6.009)

11.18 (4.539)

4.34 (6.029)

Day 9-12

16.02 (5.296)

16.31 (5.531)

11.33 (8.446)

12.65 (4.974)

Day 12-15

17.03 (6.807)

19.04 (4.473)

21.84 (9.577)

19.03 (6.453)

Day 15-18

43.52 (9.483)

41.27 (5.755)

37.83 (16.192)

44.11 (9.902)

Day 18-21

51.61 (15.190)

57.79 (8.681)

63.34 (11.295)

57.30 (12.247)

Day 6-15

44.59 (12.727)

44.47 (9.565)

44.35 (9.870)

36.02 (7.850)

Conclusions:
In rats, the maternal NOAEC was 3000 ppm (10560 mg/m3), and the maternal LOAEC was 9000 ppm (31680 mg/m3) based on color changes in the lungs, reduced body weight gain, and reduced food consumption. The developmental NOAEC 9000 ppm (31680 mg/m3) in rats.
Executive summary:

The purpose of this study was to examine the developmental toxicity of commercial hexane in rats. Groups of 25 pregnant female rats were exposed to concentrations of 0, 900, 3000, or 9000 ppm for 6 hrs/day during gestational days 6 -15. The animals were then sacrificed on GD 21. During the study, the animals were examined for clinical signs, mortality, food and water consumption, and body weights taken. After sacrifice, the internal organs were examined, and the uterus was examined for viable fetuses, number of resorptions, and number of corpora lutea. Fetuses were examined for malformations. Necropsy revealed color changes in the lungs of females in the 9000 ppm groups along with reduced body weight gain, and reduced food consumption. No treatment related abnormalities was seen in the fetuses. The maternal NOAEC in rats was 3000 ppm (10560 mg/m3), and the LOAEC 9000 ppm based on lung color changes, reduced body weight gain, and reduced food consumption. The developmental NOAEC in rats was 9000 ppm (31680 mg/m3).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report equivalent or similar to OECD guideline 414
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Exposure from GD 6-15 and rats sacrificed on GD 21
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Source - Charles River Breeding Labs, Inc. Wilmington, Mass 01887
Age at receipt - 7 weeks
Age at mating - 9 weeks (females)
Food - Purina lab chow ad libitum expect during 6-hour exposure period
Water - Ad libitum except during 6-hour exposure period

Temperature - monitored twice daily
Light cycle - 12/12 light/dark cycle
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Dry air at room temperature was drawn directly through the one cubic meter exposure chamber. Animals were exposed for 6 hours each day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Charcoal traps were removed from effluent line and transfered to scintillation-type containers. Samples were collected from two dose levels at various intervals during the exposure period (day 0 - 5 hr, day 1 - 1 hr, day 2 - 3 hr, day 3 - 5 hr, day 4 - 1 hr, week 3 - 5 hr and week 6 - 1 hr). Samples were desorbed using tetrahydrofuran, injected into a gas chromatograph for evaluation against a previously injected standard of MRD-78-26. Results of the evaluation period demonstrated that no appreciable change occurred in the distribution of the components of the test article in the vapor phase.
Details on mating procedure:
Females selected for mating were placed with males nightly in a 1:1 ratio. Vaginal smears were taken early in the morning and females were considered to have mated if sperm and/or vaginal plug were observed. Day on which evidence of mating was observed was defined as Day 0 of gestation.
Duration of treatment / exposure:
Gestation days 6-15
Frequency of treatment:
6 hours/day
Duration of test:
Gestation days 0-21
Remarks:
Doses / Concentrations:
0, 400 or 1200 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
20 females/group
Control animals:
yes, sham-exposed
other: Positive control - 400 mg/kg Acetylsalicylic acid
Details on study design:
20 pregnant female rats were included as a positive control. Rats were dosed orally with 400 mg/kg/day acetylsalicylic acid (ASA) on gestation days 6-15. Appropriate amounts of acetyl salicylic acid (ASA) were suspended in 0.5% Methocel® and administered (daily as a single dose) at a constant volume of 10 ml/kg/day. Individual doses were adjusted by daily body weights taken on Days 6-15 of gestation.
Maternal examinations:
For mortality and gross evaluations - Twice daily
Detailed physical examinations for sings of local or systemic toxicity - Days 0, 6-15 and 21 of gestation
Body weight - Measured on days 0, 6-15 and 21 of gestation
Body weight change - calculated for days 0-6, 6-15 and 15-21 of gestation
Ovaries and uterine content:
Number and location in each uterine horn of live/dead fetuses, late/early resorptions and implantation sites were evaluated. Number of corpora lutea per ovary were assessed.
Fetal examinations:
Gross dissection and examination of viscera (including internal sex determination). Skeletons were examined for malformations and ossification variations after Alizarin red staining using a modified Crary method. Approximately 1/3 of fetuses were fixed with Bouin's stain and examined for neural and visceral defects after being serially sectioned by the slicing technique of Wilson.
Statistics:
Comparisons between control Group I and control Group II, and between control Group I and each test substance-treated groups were made where applicable (incidence data) by the chi-square method or by the F-test and Student's t-test (absolute data). When variances differed significantly. Student's t-test was appropriately modified using Cochran's approximation (t1). Mean number of live fetuses, resorptions, implantations and corpora lutea were compared to control by the one-tailed t-test.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects on mortality, mean body weight gain and pregnancy rate compared to chamber controls. Physical evaluation revealed no indication of a treatment-related effect. Mean number of corpora lutea, mean numbers of implantation sites, implantation efficiency, mean number of live fetuses, resorption sites and incidence of dams with one or more resorption sites were comparable between treatment and chamber-exposed control groups. Necropsy evaluations revealed no treatment-related effects.
Dose descriptor:
NOAEC
Effect level:
>= 1 200 ppm (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean fetal weights and mean crown-rump distances in both sexes were comparable to chamber controls. Incidence of fetuses with at least one ossification variation was significantly higher in the 1200 ppm group compared to chamber controls but the incidence of litters containing fetuses with ossification variations, types and incidences of ossification variations noted where generally similar between control and test substance-treated groups.
Fetal examinations revealed no malformations in any of the treated groups.
Dose descriptor:
NOAEC
Effect level:
>= 1 200 ppm (nominal)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The NOAEC for developmental toxicity was 1200 ppm (approximately 5500 mg/m3), the highest concentration tested.
Executive summary:

A Segment II teratology study on Hydrocarbons, C7-C9, isoalkanes (CAS RN 90622-56-3), showed no evidence of embryonic or teratogenic effects in rats. In this study, pregnant rats were exposed to 0, 400, or 1200 ppm for 6 hours per day during gestation days 6 to 15. There was no mortality and no treatment-related effects to the dams. No treatment-related effects were observed for the number of live fetuses, fetal size, sex distribution, and external soft-tissue or skeletal examinations. Under the conditions of the study, there was no evidence of embryotoxicity or teratogenicity. The NOAEC for developmental toxicity was 1200 ppm (approximately 5500 mg/m3), the highest concentration tested.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
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 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
Remarks:
Doses / Concentrations:
100 ppm
Basis:
nominal conc.
525 mg/m3
Remarks:
Doses / Concentrations:
300 ppm
Basis:
nominal conc.
1575 mg/m3
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.
Dose descriptor:
NOAEC
Effect level:
>= 1 575 mg/m³ air (nominal)
Basis for effect level:
other: developmental 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.
Dose descriptor:
NOAEC
Effect level:
>= 1 575 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
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.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1978
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:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Conducted according to the Food and Drug Administration 1966 "Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use", Segment II (Teratological Study)
GLP compliance:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Bredding Laboratories
- Age at study initiation: females (58 days); males (sexually mature)

- Housing: individually except during mating
- Diet (e.g. ad libitum): ad libitum (food removed during exposure period)
- Water (e.g. ad libitum): ad libitum (water removed during exposure period)


Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
Appropriate amounts of test material were transferred from a reservoir using a metering pump into a heated flask and flash evaporated. A stream of clean air was also passed through the flask and the vapor laden air transferred to a port in the chamber air inlet, where it was diluted with normal chamber intake air to give the desired concentration. Adjustments in the exposure air concentration were made by changing the rate of the flow of test material through the metering pump.

The stainless steel and glass exposure chambers and an effective exposure volume of 760 liters. They were operated dynamically at a flow rate of approximately 125 liters per minute. This provided one air change every 8 minutes and a 99% equilibrium time of 39 minutes.

Atmospheric sampling was performed using a Wilks Scientific Corp Miran IA Ambient Air Analyzer (long pathlength infrared). The infrared spectrum of the test material was measured and a strong band associated with the test material was observed at 3.4 microns. Calibration curves relating the absorption at this wavelength to the airborne concentration of the test materials were prepared. On each exposure day, three samples were drawn from each exposure chamber and the exposure concentration calculated by comparing the absorption of this sample to the standard curve.

Postive control animals were treated via gastric intubation on gestational days 6-15 with 400mg/kg/day of acetylsalicylic acid in 0.5% methocel.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
All females selected for mating were places with male rats nightly in a 2:1 ratio. Vaginal smears were taken early in the morning and females were considered to have mated if sperm and/or a vaginal plug were observed. The day on which evidence of mating was first observed was established as Day 0 of gestation for that animal. Mated females were assigned to groups by daily body weight gain in an attempt to equalize Day 0 mean group body weights.
Duration of treatment / exposure:
Females were exposed on gestation days 6-15 by inhalation 6h/day
Frequency of treatment:
daily gestation days 6-15
Duration of test:
Day 6 of gestation ranged from 23 January-3 February 1978
Day 15 of gestation ranged for 1-12 February 1978
Remarks:
Doses / Concentrations:
300 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
900 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
Negative control (Chamber air)- 20 mated females
Postive control (acetylsalicylic acid)-20 mated females
300 ppm- 21 mated females
900ppm- 21 mated females

Control animals:
yes, sham-exposed
other: positive control treated with 400mg/kg/day acetylsalicylic acid
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


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



POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: uterus (number and location recorded for each horn of the following: live fetuses, dead fetuses, late resorptions, early resorptions, implantation sites); ovaries (number of corpora lutea per ovary)


Fetal examinations:
All fetuses were weighted, crown-rump distance measured, examined externally for malformations and sex determined externally (anogenital distance)
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes
- Skeletal examinations: Yes: [2/3 of litter ]
Fetuses designated for skeletal evaluation were eviscerated prior to initiation of the skeletal staining procedure. During the evisceration step the visceral contents of the thoracic and abdominal cavities were evaluated grossly in situ and sex was determined by internal inspection of gonads. Examination of skeleton for anomalies and ossification variations was performed after staining.
- Neural and Visceral defects: Yes: [1/3 of litter]
Statistics:
Comparisons between the negative control and treated groups and between the negative control and positive control groups were made where applicable by the chi-square method. Body weights, body weight gains, numbers of corpora lutea, implantations, resorptions, fetuses per dam, fetal and litter weights and crown-rump distances were compared to control by the F-test and Student’s t-test. When variances differed significantly, Student’s t-test was appropriately modified using Cochran’s approximation.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Animals treated with 900 ppm exhibited a slight increase in excessive lacrimation during the treatment and post-treatment periods. This same group also exhibited an increased incidence of brown flakes in the fur covering the head area during the treatment period. Premature delivery of the litter on Day 21 of gestation prior to maternal sacrifice was observed in one negative control female, and two test material treated females. There were no remarkable gross postmortem changes in the treated adult females. All other physical observations occurred with similar frequencies in all groups and were considered to represent common observations noted in rats in the laboratory environment.

Positive control animals demonstrated statistically significant decreased body weight gain. Females had in utero litters containing fewer live fetuses and more resorption sites than untreated control litters. The implantation efficiency value was significantly reduced and the incidence of dams with two or more resorptions was increased.
Dose descriptor:
NOAEC
Effect level:
>= 5 220 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
All fetal survival, size and sex data for groups treated with test material were considered comparable to negative control data. Slight delays or variation in the normal ossification process were observed in treated animals. However such variation are common as the time of normal ossification can vary and were comparable to the variation observed in the control animals. The incidence of fetuses with external malformations and incidences of litters containing malformed fetuses in the groups treated with test material were considered comparable to the control data. No significant difference in the incidence of visceral malformations was observed in the treated groups. The incidence of fetuses with soft tissue malformation in groups treated with test material was comparable to the negative control.

In the positive control group, the percentage of live fetuses and mean fetal size data were significantly lower than the negative control and the percentage of resorbed fetuses was significantly higher than control. The incidence of fetuses with ossification variation was significantly higher than the control value. The incidence of fetuses with soft tissue malformations was significantly higher in the positive control treated group than the negative control.
Dose descriptor:
NOAEC
Effect level:
>= 5 220 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Abnormalities:
not specified
Developmental effects observed:
no
Conclusions:
There was no evidence of maternal or fetal toxicity at either exposure level of MRD-77-44 tested. Based on these results, both the maternal and developmental NOAELs were greater than or equal to 900 ppm (>= 5220 mg/m^3).
Executive summary:

MRD-77-44 was administered to pregnant female rats by inhalation exposure to vapor concentrations of 300 or 900 ppm, 6 hours/day during gestation days 6 to 15 to assess developmental toxicity.  Included in this study was a negative control (chamber exposed) group and a positive control group that was treated via gastric intubation on gestational days 6-15 with 400mg/kg/day of acetylsalicylic acid.  All surviving females were sacrificed on Day 21 of testation and fetuses examined for external, soft tissue and skeletal malformations.  Pregnancy rate, mortality, body weight gain and gross postmortem observations were unaffected by treatment.  MRD-77-44 treatment at either dose level had no effect on reproductive endpoints, fetal size, sex distribution, ossification variation, or fetal examination endpoints.    Thus, there was no evidence of maternal or fetal toxicity at either exposure level of MRD-77-44 tested.  Based on these results, both the maternal and developmental NOAELs were greater than or equal to 900 ppm (5220 mg/m3).

Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Additional information

One developmental study was available in rats orally exposed to C16-C20 n-alkanes, isoalkanes, cyclics, <2% aromatics showing a NOAEL>1000 mg/kg/day for both maternal and developmental toxicity.

READ ACROSS DATA: C16 to C30 highly purified light mineral oil (60% paraffins, 40% naphthenes)

A series of studies were conducted to evaluate the teratogenic effects of a C16-C30 light mineral oil in pregnant Sprague-Dawley rats by three separate routes of exposure; inhalation (1000 mg/m3), dermal (2000 mg/kg bw/day), oral gavage (5000 mg/kg bw/day) with exposures on gestation days 6-19. Due to low vapor pressure, the mineral oil was delivered as an aerosol for the inhalation study though aerosol particle size generated was well within the respirable range. There were no observable maternal effects (food consumption, body weight gain) irrespective of route of exposure. No adverse effects on reproductive parameters (implantation number, resorptions, fetal viability) or fetal parameters (body weight, crown-rump length) were observed in any of the studies. There was a clear lack of teratogenicity with regard to abnormal ossification, and adverse effects to external or soft tissue. Spontaneous anomalies were noted in some exposure groups but were not considered exposure related since incidence of such occurrences were similar in the sham-exposed groups. NOAEL for developmental toxicity was ≥ 2000 mg/kg bw/day (dermal), 1000 mg/m3 (inhalation) and 5000 mg/kg bw/day (oral gavage).

Justification for classification or non-classification

These findings do not warrant the classification of C14-C20 aliphatics, <2% aromatic hydrocarbons as teratogenic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under the Directive 67/548/EEC for dangerous substances.