Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available (further information necessary)
Additional information

No guideline studies were located that have examined the potential impact on reproductive function. Some indication of the likely effect of a test substance on reproductive organs can be gained from the results of repeated-dose toxicity studies with closely related materials, as summarized in Table 1.

 

Table 1. Summaries of data on reproductive organs from subchronic studies with related substances. (Robust study summaries are provided in Section 7.5 Repeated Dose Toxicity)

Test Material

Route, Species, Doses, Exposure Regimen

Endpoints

Results

Reference

Diesel fuel

Inhalation. Rat.

350, 880, 1710 mg/m3

4 hr/day, twice/wk, 13 wk

Weight and histopathology of testes

No treatment-related effect noted

Lock et al, 1984

Cherry point diesel fuel No. 2

CAS 68476-34-6

Dermal. Rat.

0.5, 2.0, 5.0 ml/kg

5 d/wk, 4 wk

Weight and histopathology of testes and ovaries

No treatment-related effect noted

ARCO, 1986a

Naval distillate Watson

CAS 68334-30-5

Dermal. Rat.

0.25, 2.0, 5.0 ml/kg

5 d/wk, 4 wk

Weight and histopathology of testes and ovaries

No effect noted. Testicular degeneration in 2/10 males considered spontaneous.

ARCO, 1986b

Watson diesel fuel #2

68476-34-6

Dermal. Rat.

0.5, 2.0, 5.0 ml/kg

5 d/wk, 4 wk

Weight and histopathology of testes and ovaries

No treatment-related effect noted

ARCO, 1986c

Diesel fuel

CAS 68476-34-6

Dermal. Rat.

0.5, 1.0, 2.5, 5.0, 10.0 ml/kg

5 d/wk, 4 wk

Weight and histopathology of testes and ovaries

No treatment-related effect noted

ARCO, 1988

Naval distillate

CAS 68334-30-5

Dermal. Rat.

0.0001, 0.005, 0.5 ml/kg

5 d/wk, 4 wk

Weight and histopathology of testes and ovaries

No treatment-related effect noted

ARCO, 1992a

Naval distillate

CAS 68334-30-5

Dermal. Rat.

0.50 ml/kg

5 d/wk, 4 wk

Weight of testes and ovaries. No histopathology was done.

No treatment-related effect noted

ARCO, 1994a

Diesel fuel

(F-237)

68334-30-5

Dermal. Rat.

0.01, 0.10, 1.00 ml/kg,

5 d/wk, 13 wk

Weight of testes and ovaries. Histopathology of testis, ovary, prostate, uterus

No treatment-related effect noted

ARCO, 1994b

Based on test results from the above studies, it is considered unlikely that exposure will affect reproductive performance. However an extended one-generation reproductive toxicity test (OECD 443) has been proposed by Concawe to fulfil the data requirement for the VHGC Category, which is still under consideration. As this Category contains substances that are structurally related to the registered material it is proposed that the reproductive toxicity endpoint will be fulfilled by read-across to the Concawe study once complete.


Short description of key information:
No guideline studies were identified for effects on reproductive function.
Gonadal histopathology and/or sperm parameters (counts; morphology) were among endpoints included in a subchronic study with aerosol inhalation exposure to diesel fuel. No effects on reproductive organs were observed and the NOAEL was 1710 mg/m3 (for aerosolised diesel fuel).
An extended one generation reproduction toxicity test (OECD 443) is proposed to fulfil the data requirement but the need for this study for this is questioned based on the fact that there were no effects observed on reproductive organs in repeated dose toxicity tests or in prenatal developmental tests. Overall it is considered unlikely that exposure will affect reproductive performance.

Effects on developmental toxicity

Description of key information

Results of two inhalation developmental toxicity studies indicate a NOAEC >2,110 mg/m3. Maternal and foetal NOAELs of 125 mg/kg body weight/day were established from dermal prenatal developmental toxicity studies (equivalent or similar to OECD 414). In the dermal studies the only fetal effect was reduced pup weight, which occurred at dose levels that also caused maternal toxicity. There are no acceptable developmental studies following oral exposure.

Additional testing is now proposed by Concawe to fulfil the data requirement for the VHGO Category, dermal tests to be conducted in two species (rat and rabbit). As this Category contains substances that are structurally related to the registered material it is proposed that the developmental toxicity endpoint will be fulfilled by read-across to the Concawe studies once complete.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1978-11-13, end date not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions because it was well documented and performed similar to OECD 414. No information on GLP.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
There were few details about the test compound.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: CRL:COBS CD (SD)BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Massachusetts
- Age at study initiation: 11 weeks old
- Weight at study initiation: Not reported
- Housing: Individually except during mating
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum, acidified (pH 2.5) water
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light


IN-LIFE DATES: From:1978-11-13 To: Not reported
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: Warm glass wool filled flask with compressed air
- Method of holding animals in test chamber: Stainless steel and plexiglass containers
- Source and rate of air: Compressed air at an unspecified rate
- Method of conditioning air: Not reported
- Temperature, humidity, pressure in air chamber: Under negative pressure at the following temperatures: control=26.7+/-1.04 degrees Celsius; 100 ppm=25.5+/-1.30 degrees Celsius; 400 ppm=26.8+/-1.00 degrees Celsius
- Air flow rate: 28.3 litres per minute
- Air change rate: Not reported
- Method of particle size determination: Not determined
- Treatment of exhaust air: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Scott Model 216 Hydrocarbon Analyzer
- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were monitored hourly using a Scott Model 216 Hydrocarbon Analyzer calibrated with known concentrations of diesel fuel. Methane was used as an internal standard.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: Not reported
- Length of cohabitation: Not reported
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
10 days (gestational days 6 through 15)
Frequency of treatment:
6 hours a day
Duration of test:
14 days
No. of animals per sex per dose:
Twenty pregnant dams per dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Stated as selected by API.
- Rationale for animal assignment (if not random): Assigned sequentially to the dose groups

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations included general appearance, behaviour and condition.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: On gestational days 0, 6, 15, and 20


FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No



WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Visceral and thoracic organs
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- 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: one-third per litter
- Skeletal examinations: Yes: two-thirds per litter
- Head examinations: Yes: one-third per litter
Statistics:
Continuous parameters were analyzed with Dunnet's t-test. A 2x2 contingency table with Yates correction was used for ratio data. Discontinuous data (e.g., number of abnormal foetuses per litter) were analyzed using Wilcoxon Rank Sum test.
Indices:
Nidation index, liver and dead foetuses, implantations, pup weight, sex ratio, and abnormalities
Historical control data:
Historical data on 54 litters was provided for reproductive performance. The concurrent control was considered to be similar to the historical control.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no deaths during the course of the study and all females were normal in appearance. Mean body weights indicated no significant differences between control and treated pregnant rats, although the food consumption of the high-dose group was significantly lower (p < 0.05) than the controls during treatment. Examination at necropsy revealed two high-dose females with dark, mottled lungs. One of these animals had a rough spleen but no further description was provided for this and the authors did not consider either of these effects to be treatment related.

Key result
Dose descriptor:
NOAEC
Effect level:
401.5 ppm (analytical)
Based on:
other:
Remarks:
overall effects
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Examination of the offspring revealed no visible abnormalities except for the occurrence of subcutaneous haematomas in 1/188, 4/227 and 4/188 in the control, low-, and high-dose groups, respectively. All litters appeared normal and the sex ratio did not differ significantly between treated and control groups. Some unusual skeletal variations related to retarded bone ossification were observed, including reduced ossification of the parietal bones and the frontal bones, and wavy ribs. These changes were noted in 7 and 17 pups from 3 and 8 litters of the control and low-dose animals only, and were not dose related. The authors concluded that neither the frequency nor the character of these changes indicated an adverse effect on foetal growth and development or a teratogenic potential. Other skeletal variations were considered to be common to animals of this strain.
Key result
Dose descriptor:
NOAEC
Effect level:
401.5 ppm (analytical)
Based on:
other:
Remarks:
overall effects
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Exposure of pregnant rats to diesel fuel (vapour) did not result in compound-induced maternal toxicity, teratology, variation in sex ratio, embryo toxicity or inhibition of foetal growth and development. Therefore the NOAEC was 2,110 mg/m3, the highest concentration tested.
Executive summary:

The teratology of diesel fuel was examined by whole body exposure of pregnant female (CRL: COBS CD (SD) BR) rats to graded measured airborne (vapour) of 0, 101.8, or 401.5 ppm (equivalent to 0, 530, and 2110 mg/m3, respectively) on days 6 through to 15 of gestation. The pregnant female rats were approximately 11 weeks of age at the time of the first dose and were assigned sequentially to treatment groups of 20 animals each. They were exposed to diesel fuel for 6 hours/day in 0.25 m3 stainless steel and plexiglass chambers operated under negative pressure. The animals were assessed daily for body weight changes, food consumption, and clinical observations.

There were no deaths during the course of the study and all females were normal in appearance. Mean body weights indicated no significant differences between control and treated pregnant rats, although the food consumption of the high-dose group was significantly lower (p < 0.05) than the controls during treatment.

On day 20 of gestation, the study was terminated and the rats were anaesthetised with chloroform. Following anaesthesia, gross examination of the organs, examination of the uterus (number of implantation sites, live/dead foetuses, number of resorption sites) and foetal examinations (soft tissue and skeletal) were performed.

Examination at necropsy revealed two high-dose females with dark, mottled lungs. One of these animals had a rough spleen but no further description was provided for this and the authors did not consider either of these effects to be treatment related.

Diesel fuel did not appear to have any effect on the uterus and examination of the offspring revealed no visible abnormalities except for the occurrence of subcutaneous haematomas in 1/188, 4/227 and 4/188 in the control, low-, and high-dose groups, respectively. All litters appeared normal and the sex ratio did not differ significantly between treated and control groups. Some unusual skeletal variations related to retarded bone ossification were observed, including reduced ossification of the parietal bones and the frontal bones, and wavy ribs. These changes were noted in 7 and 17 pups from 3 and 8 litters of the control and low-dose animals only, and were not dose related. The authors concluded that neither the frequency nor the character of these changes indicated an adverse effect on foetal growth and development or a teratogenic potential. Other skeletal variations were considered to be common to animals of this strain.

 

The authors concluded that exposure of ratsto diesel fuel (vapour) did not result in compound-induced terata, variation in sex ratio, embryo toxicity or inhibition of foetal growth and development. Therefore the NOAEC was 2,110 mg/m3 (401.5 ppm), the highest concentration tested.

This study received a Klimisch score of 2 and is classified as reliable with restrictions because the study is well-documented and generally followed OECD 414 guidelines. No information was available on GLP.

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because the high-dose had to be discontinued due to maternal discomfort and was not available to determine developmental toxicity, but the study was well conducted and was GLP compliant.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
No skeletal or visceral exams were conducted.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
dermal
Vehicle:
acetone
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
20 days
Frequency of treatment:
daily
Duration of test:
20 days
No. of animals per sex per dose:
between 14 and 19 females
Control animals:
yes, concurrent vehicle
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
dermal irritation, reduced body weight gain
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
reduced pup weight post parturition until day 4
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The maternal LOAEL is 125 mg/kg/day, based on dermal irritation. There is no maternal NOAEL. The developmental LOAEL is 125 mg/kg/day, based on reduced pup weight on lactational day 0. There is no developmental NOAEL. 
Executive summary:

In a developmental toxicity study, VDF diesel in acetone was dermally administered to 14 to 19 presumed pregnant rats/dose at dose levels of 0, 125, or 250 mg/kg bw/day from days 0 through 20 of gestation. Another group of 14 received 1000 mg/kg/day from days 0 to 5 of gestation.

 

Dermal irritation occurred in all treatment groups. Body weight, body weight gain, and food consumption was reduced in the 250- and 1000 -mg/kg/day groups. The maternal LOAEL is 125 mg/kg/day, based on dermal irritation. There is no maternal NOAEL. Pup weight was decreased on lactational day 0 in all dose groups and on lactational day 4 in the 250 -and 1000 -mg/kg/day groups. The developmental LOAEL is 125 mg/kg/day, based on reduced pup weight. There is no developmental NOAEL.

 

This study received a Klimisch score of two and is classified as reliable with restrictions because the high-dose had to be discontinued due to maternal discomfort and was not available to determine developmental toxicity, but the study was well conducted and was GLP compliant.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Concawe believes that dermal is the most relevant exposure route, and is sufficiently robust, to identify any potential hazards from repeated exposures to petroleum products to be able to adequately manage the potentially associated risks. Please see attached document for further justification.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Ultra-Low Sulfur Diesel Fuel
CAS 68334-30-5
WIL sample ID no. 110129
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: 79 days old upon receipt; at initiation 91 days
- Housing: until pairing, all rats were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board; rats were paired for mating in the home cage of the male.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, available ad libitum
- Water (e.g. ad libitum): Municipal water supply, available ad libitum
- Acclimation period: 12 days, during which time the animals were acclimated to wearing Elizabethan-style collars on an incremental basis starting on study day -8 (1 hour), -7 (4 hours), -6 (8 hours), -5 (24 hours)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 71°F ± 5°F (22°C ± 3°C); Actual mean daily temperature ranged from 70.9°F to 77.2°F (21.6°C to 25.1°C)
- Humidity (%): 50% ± 20%; Actual mean daily relative humidity ranged from 38.0% to 50.4%
- Air changes (per hr): minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod

IN-LIFE DATES: From: To: 15th September 2011 to 20th October 2011
Route of administration:
dermal
Vehicle:
other: mineral oil
Remarks:
USP (lot no. 2AF0729, exp. date: 3 August 2013, manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ
Details on exposure:
TEST SITE
- Area of exposure: dorsal scapular area
- % coverage: 10%
- Type of wrap if used: no wrapping - Elizabethan collar worn to minimize ingestion of test substance
- Time intervals for shavings or clipplings: the day prior to initiation of dose administration and as often as necessary thereafter (either prior to dosing or 2-4 hrs after the 6 hr exposure period).

REMOVAL OF TEST SUBSTANCE
- Washing (if done): patted with paper towel (if necessary a gauze moistened with mineral oil could be used followed by a dry paper towel)
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.5 mL/kg
- Concentration (if solution): 0, 66.7, 200, 400 mg/mL for control, 100, 300, 600 mg/kg/day groups respectively
- Constant volume or concentration used: no - Individual dosages and dose volumes were based on the most recently recorded body weights to provide the correct mg/kg/day dose

VEHICLE
mineral oil, USP (lot no. 2AF0729, exp. date: 3 August 2013, manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ)

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes - Elizabethan collars
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A validated GC/MS method was used for the determination of test substance concentration in suspension formulations. Duplicate samples from the top, middle, and bottom strata of the formulations prepared on 26 September 2011 and 12 October 2011 at target test substance concentrations of 66.7, 200, and 400 mg/mL were analyzed to assess test substance homogeneity/concentration acceptability. Duplicate samples from the blank vehicle were also collected and analyzed. The analytical results met the applicable WIL Research SOP acceptance criteria for test substance homogeneity/concentration acceptability with the following exception. The results of the initial assessment of the 26 September 2011 Group 3 (66.7 mg/mL) formulation failed to meet the acceptance criteria. A processing error was suspected to have occurred. The back-up samples were analyzed, and the overall mean concentration percent of target was reported as 89.2%, which met the previously stated WIL Research SOP requirements for test substance homogeneity. No test article was detected in the analyzed vehicle administered to the control group.

Homogeneity, stability, and resuspension homogeneity of the test substance formulations were previously established at concentrations of 50 and 500 mg/mL following 8 days of room temperature storage during a preliminary study. Therefore, stability and resuspension homogeneity analyses were not conducted during the current study.
Details on mating procedure:
- Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]
- M/F ratio per cage: 1:1; females housed in the home cage of males of the same strain
- Proof of pregnancy: presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist referred to as gestation day 0
Duration of treatment / exposure:
6 hours
Frequency of treatment:
Daily
Duration of test:
Gestation days 0-19
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Sham exposure and vehicle controls
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
This was the highest tolerable dose (due to irritation seen in the range-finding study for a 90-day dermal OECD 411 study at 1000 mg/kg/day)
No. of animals per sex per dose:
25 pregnant rats per group
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Details on study design:
The experimental design consisted of 3 test substance-treated groups, 1 vehicle control group, and 1 sham control group composed of 25 rats per group
- Dose selection rationale:
- Rationale for animal assignment (if not random): bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon
Individual clinical observations were recorded from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity at the time of dose administration and approximately 1-2 hour following dose administration. The absence or presence of findings was recorded for individual animals

DERMAL OBSERVATIONS: Yes
- Time schedule: daily
The application site was scored daily (prior to dose administration) during the treatment period for erythema and edema. Other remarkable dermal findings, if present, were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights recorded on gestation days 0, 3, 6, 9, 12, 15, 18 and 20. Group mean weights and weight changes also calculated. Elizabethan collars removed for weighing.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual food consumption was recorded on gestation days 0, 3, 6, 9, 12, 15, 18, and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 by CO2 inhalation
- Organs examined:
The cranial, thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined
The uterus and ovaries were then exposed and excised (see below).
The liver, brain, and thymus were weighed from all females. Organ to brain weight ratios were calculated. The following tissues and organs were collected and placed in
10% neutral-buffered formalin:
Treated skin
Brain
Untreated skin (right hindlimb)
Thymus
Liver (2 sections)
All gross lesions

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
- Other:
All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.

Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss

The placentae were also examined
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]

Each viable fetus was examined externally, individually sexed, weighed. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.

Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels.

The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development.

Heads from approximately one-half of the fetuses in each litter were placed in Bouin's fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the
fetuses were examined by a midcoronal slice.

All carcasses were eviscerated and fixed in 100% ethyl alcohol.

Following fixation in alcohol, each fetus was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the vehicle control group (Group 2). In addition, the vehicle control group (Group 2) was compared separately to the sham control group (Group 1). Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses.

Mean maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, fetal body weights (separately by sex and combined), and organ weights were subjected to a parametric one-way ANOVA.

If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test or a two-sample t-test, as appropriate, was used to compare the test substance-treated groups to the vehicle control group and the vehicle control group to the sham control group.

Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test.

If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the vehicle control group and the vehicle control group to the sham control group.
Indices:
1. Group Mean Litter Basis:
Postimplantation Loss/Litter = No. Dead Fetuses,Resorptions (Early/Late)/Group {divided by} No. Gravid Females/Group

2. Proportional Litter Basis:
Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%) {divided by} No. Litters/Group

Where:
Postimplantation Loss/Litter (%) = No. Dead Fetuses, Resorptions (Early/Late)/Litter {divided by} No. Implantation Sites/Litter {times} 100
Historical control data:
WIL Research has historical control data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Findings noted (including yellow, red and/or brown material in the urogenital and ventral abdominal areas and hair loss on vaious body surfaces occurred infrequently and at similar frequencies across all test and control groups. They were likely due to the animals' inability to groom and discomfort caused by the Elizabethan collar and not attributed to test substance administration.
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
No remarkable dermal observations were noted in the 100, 300, and 600 mg/kg/day groups. Observations in these groups occurred infrequently, at similar frequencies in the vehicle control group, and/or in a manner that was not dose-related.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100, 300, and 600 mg/kg/day groups were unaffected by test substance administration. Differences from the vehicle control group were slight and not statistically significant.
Although some differences between sham and vehicle controls that achieved statistical significance were noted (lower bodyweights in vehicle control), these were attributed to biological variability.

See Table 1 below in "Any other information on results" section
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, and 600 mg/kg/day groups was unaffected by test substance administration. Differences from the vehicle control group were slight and not statistically significant.
Mean food consumption in the vehicle control group was higher than sham control on some gestation days. However, because there was no corresponding effect on mean body weight parameters in the vehicle control group, as mean body weight gains in the vehicle control group were often lower than the sham control group, these changes were attributed to biological variability.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
no behaviour related abnormalities were noted as part of the general clinical observations
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test substance-related effects on organ weights (absolute and relative to brain weight) were observed in any treatment group. Differences from the vehicle control group were slight and not statistically significant. Mean organ weights (absolute and relative to brain weight) in the vehicle control group were generally similar to the sham control group. Differences from the sham control
group were slight and not statistically significant with the following exception. A significantly (p<0.05) lower mean thymus weight was noted in the vehicle control group compared to the sham control group. This difference was attributed to biological variability.

See Table 1 below in "Any other information on results" section for gravid uterine weights
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose-related. No remarkable macroscopic findings were noted in the vehicle control group compared to the sham control group. Findings noted in the vehicle control group were limited to single females.

3, 4, and 3 females in the sham control, vehicle control, and 300 mg/kg/day groups, respectively, were determined to be nongravid.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
At the scheduled necropsy no test substance-related internal findings were observed at any exposure level.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Early or late resorptions:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Dead fetuses:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Details on maternal toxic effects:
Based on the lack of maternal toxicity, the no-observed-adverse-effect level (NOAEL) was determined to be 600 mg/kg/day (the highest dose level evaluated)
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects at top dose tested
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for morphological evaluation were 334(22), 302(21), 368(25), 330(22), and 374(25) in the sham control, vehicle control, 100, 300, and 600 mg/kg/day groups, respectively. Malformations were observed in 1(1), 2(2), 1(1), 0(0), and 0(0) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.

No test substance-related external malformations were noted for fetuses in the test substance-treated groups. One fetus in the 100 mg/kg/day group was observed with tarsal flexure (right). There was no apparent skeletal origin for this finding. This external malformation was not considered test substance-related as it did not occur at higher exposure levels (300 or 600 mg/kg/day).

See attached document "402014 Fetal malformation summaries" for more details
Skeletal malformations:
no effects observed
Description (incidence and severity):
Malformations
There were no skeletal malformations in the test substance-treated groups.

Variations
Skeletal developmental variations observed in the test substance-treated groups consisted of hyoid unossified, sternebra(e) nos. 5 and/or 6 unossified, cervical centrum no. 1 ossified, sternebra(e) malaligned (slight or moderate), reduced ossification of the 13th rib(s), reduced ossification of the vertebral arches, 14th rudimentary rib(s), 7th cervical rib(s), 27 presacral vertebrae, unco-ossified vertebral centra, 25 presacral vertebrae, sternebra(e) nos. 1, 2, 3, and/or 4 unossified, reduced ossification of the skull, and bent rib(s). These findings occurred infrequently or at a frequency similar to the vehicle control group, did not occur in a dose-related manner, and/or the mean litter proportions were within the WIL historical control data range.

See attached document "402014 Fetal malformation summaries" for more details
Visceral malformations:
no effects observed
Description (incidence and severity):
Malformations
No visceral malformations were noted in the test-substance treated groups.

Variations
A major blood vessel variation (right carotid and subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk]) was noted for 1 fetus each in the 100 and 300 mg/kg/day groups. This visceral developmental variation was not considered to be test substance-related as it occurred in single fetuses and in a manner that was not dose-related.
Distended ureters were noted for 3 fetuses from 1 litter in the 300 mg/kg/day group and 4 fetuses from 1 litter in the 600 mg/kg/day group. The mean litter proportions of this finding in these groups (1.4% and 1.1% per litter, respectively) were not statistically significantly different from the vehicle control group (0.0% per litter) and the values were within the range of values in the WIL historical control data (0.0% to 4.0% per litter). Therefore, this finding was not considered test article-related.

See attached document "402014 Fetal malformation summaries" for more details
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Based on the lack of toxicity or effects on intrauterine growth and survival and fetal morphology at any exposure level tested, the no-observed-adverse-effect level (NOAEL) was determined to be 600 mg/kg/day (the highest dose level evaluated) for embryo/fetal developmental toxicity.
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at top dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
600 mg/kg bw/day
Treatment related:
no

Table 1: Summary of Net Body Weight Changes and Gravid Uterine Weights

Dose (mg/kg/day) Sham 0 100 300 600
Parameter          
Initial Body Wt (g) 259 ± 14 259 ± 14 260 ± 13 260 ± 17 261 ± 12
Terminal Body Wt (g) 400 ± 24 386 ± 21  389 ± 21  394 ± 33  387 ± 29
Gravid Uterus (g) 87.1 ± 9.0 82.5 ± 11.9 83.3 ± 9.5 85.3 ± 17.5 83.0 ± 11.1
Net Body Wt (g) 312 ± 20 304 ± 16 306 ± 16 308 ± 24 304 ± 21
Net Body Wt gain (g) 53.2 ± 11.3 44.9 ± 14.6*  46.1 ± 15 48.5 ± 11.7 43.5 ± 17.2

* - Test: two-sample t-test 5% significance level

Table 2: Summary of Fetal Data at Scheduled Necropsy

Dose (mg/m3) Sham 0 100 300 600
Parameter            
Number of Females Pregnant   22 21 25 22 25
Dams with Viable Fetuses   22 21 25 22 25
No. Corpora Lutea/Grp   356 337 432 382 419
No. Corpora Lutea/Animal Mean 16.2 16 17.3 17.4 16.8
SD 2 1.8 2.7 2.8 2.5
No. Implantations/Group   346 317 401 353 391
No. Implantations/Animal Mean 15.7 15.1 16 16 15.6
SD 1.9 2.5 1.5 2.2 2.2
Preimplantation Loss   10 20 31 29 28
Preimplantation loss/Animal Mean 0.5 1 1.2 1.3 1.1
SD 1.1 1.9 2.2 1.5 1.6
% Preimplantation Loss Mean 2.5 5.9 6.1 7 6.3
SD 5.9 11.6 9.3 7.9 9.1
No. of Live Fetuses/Group   334 302 368 330 374
No. of Live Fetuses/Animal Mean 15.2 14.4 14.7 15 15
SD 1.7 2.4 1.9 2.9 2.3
% of Live Male Fetuses in Litter Mean 51.5 51.4 48.1 49.1 51.9
SD 13.5 15.4 13.9 10.2 12.3
% of Live Female Fetuses in Litter Mean 48.5 48.6 51.9 50.9 48.1
SD 13.5 15.4 13.9 10.2 12.3
Postimplantation Loss   12 15 33 23 17
Postimplantation Loss/Animal Mean 0.5 0.7 1.3 1 0.7
SD 0.7 0.8 1.2 1.7 1.1
% Postimplantation Loss/Animal (%(UD)) Mean 3.3 4.7 8.3 6.7 4.3
SD 4 5.1 7.4 10.6 6.8
No. Dead Fetuses/Group   0 0 0 0 0
No. Early Resorptions/Group   12 14 33 22 17
No. Early Resorptions/Animal Mean 0.5 0.7 1.3 1 0.7
SD 0.7 0.7 1.2 1.7 1.1
% Early Resorptions/Animal Mean 3.3 4.4 8.3 6.5 4.3
SD 4 4.9 7.4 10.7 6.8
No. Late Resorptions/Group   0 1 0 1 0
No. Late Resorptions/Animal Mean 0 0 0 0 0
SD 0 0.2 0 0.2 0
% Late Resorptions/Animal Mean 0 0.3 0 0.3 0
SD 0 1.3 0 1.3 0
% Total Resorptions/Animal Mean 3.3 4.7 8.3 6.7 4.3
SD 4 5.1 7.4 10.6 6.8
Mean Male Fetal Weight (g) Mean 3.9 3.8 3.8 3.8 3.8
SD 0.28 0.23 0.25 0.3 0.31
N 22 21 25 22 25
Mean Female Fetal Weight (g) Mean 3.7 3.7 3.6 3.6 3.6
SD 0.3 0.25 0.25 0.27 0.26
N 22 21 25 22 25
Mean Combined Fetal Weight (g) Mean 3.8 3.8 3.7 3.7 3.7
SD 0.29 0.21 0.23 0.28 0.28
N 22 21 25 22 25
Conclusions:
Based on the lack of maternal toxicity or effects on intrauterine growth and survival and fetal morphology at any exposure level tested, the no-observed-adverse-effect level (NOAEL) was determined to be 600 mg/kg/day (the highest dose level evaluated) for both maternal and embryo/fetal developmental toxicity when ultra-low sulfur diesel fuel was administered by dermal application to bred Crl:CD(SD) rats.
Executive summary:

Based on the lack of maternal toxicity or effects on intrauterine growth and survival and fetal morphology at any exposure level tested, the no-observed-adverse-effect level (NOAEL) was determined to be 600 mg/kg/day (the highest dose level evaluated) for both maternal and embryo/fetal developmental toxicity when ultra-low sulfur diesel fuel was administered by dermal application to bred Crl:CD(SD) rats.

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions beacuse it was GLP compliant and was conducted generally according to OECD 414 guidelines.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD BR VAF/Plus
Route of administration:
dermal
Vehicle:
acetone
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
19 days
Frequency of treatment:
daily
Duration of test:
19 days
No. of animals per sex per dose:
25 female
Control animals:
yes, concurrent vehicle
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
decreased body weight gain, dermal irritation
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Straight run diesel did not effect embryo-fetal development or viability even with doses high enough to cause distress in the dams.
Executive summary:

In a developmental toxicity study, straight run diesel in acetone was dermally administered to 25 presumed pregnant rats/dose at dose levels of 0, 50, 150, or 300 mg/kg bw/day from days 0 through 19 of gestation.

 

Skin reactions (erythmea, oedema, atonia, and desquamation) occurred in all treatment groups. In the mid- and high-dose group, vocalization occurred in 5 or 6 of the animals. There was a slight decrease in body weight gain in the 50 mg/kg/day group with significant reductions occurring in the 150- and 300-mg/kg/day groups. Feed consumption was only reduced in the high-dose group. There were no treatment-related effects on reproduction or development (number of corpora lutea, implantations, litter size, live fetuses, resorptions, fetal bosy weight, or sex ratio). There were no gross external, skeletal, or visceral abnormalities observed. The maternal LOAEL is 50 mg/kg/day, based on dermal effects and reduced body weight gain. There is no maternal NOAEL. There is no developmental LOAEL. The developmental NOAEL is 300 mg/kg/day.

 

This study received a Klimisch score of one and is classified as reliable without restrictions because it was GLP compliant and was generally conducted according to OECD 414 guidelines.

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because the high-dose had to be discontinued due to maternal discomfort and was not available to determine developmental toxicity, but the study was well conducted and was GLP compliant.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
No skeletal or visceral exams were conducted.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Details on maternal toxic effects:
Maternal toxic effects:yes
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The maternal LOAEL is 125 mg/kg/day, based on dermal irritation. There is no maternal NOAEL. The developmental LOAEL is 250 mg/kg/day, based on reduced pup weight. The developmental NOAEL is 125 mg/kg/day.
Executive summary:

In a developmental toxicity study, straight run diesel in acetone was dermally administered to 14 or 15 presumed pregnant rats/dose at dose levels of 0, 125, or 250 mg/kg bw/day from days 0 through 20 of gestation. Another group of 15 rats received 1000 mg/kg/day from days 0 to 5 of gestation.

 

Dermal irritation occurred in all treatment groups. Body weight, body weight gain, and food consumption was reduced in the 250- and 1000 -mg/kg/day groups. The maternal LOAEL is 125 mg/kg/day, based on dermal irritation. There is no maternal NOAEL. Pup weight was decreased on lactational days 0 and 4 in the 250 -mg/kg/day group. There were no developmental effects observed in the 1000 -mg/kg/day for 6 days. The developmental LOAEL is 250 mg/kg/day, based on reduced pup weight. The developmental NOAEL is 125 mg/kg/day.

 

This study received a Klimisch score of two and is classified as reliable with restrictions because the high-dose had to be discontinued due to maternal discomfort and was not available to determine developmental toxicity, but the study was well conducted and was GLP compliant.

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1979-03-05 to 1979-09-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because it is well document and was generally conducted according to OECD 414 guidelines. No information available on GLP compliance.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Only a few details were provided on mating and exposure procedures
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: CRL:COBS CD(SD) BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: 11 weeks old
- Weight at study initiation: Not reported
- Housing: Individually in wire bottom cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): Acidified (pH 2.5) ad libitum
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Only specified as temperature controlled
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light


IN-LIFE DATES: From: 1979-03-05 To: Not reported
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: Warm flask with compressed air
- Method of holding animals in test chamber: Stainless steel and plexiglass containers
- Source and rate of air: Compressed air at an unspecified rate
- Method of conditioning air: Not reported
- Temperature, humidity, pressure in air chamber: Under negative pressure at the following temperatures: control=28.2+/-1.40 degrees Celsius; 100 ppm=27.7+/-1.36 degrees Celsius; 400 ppm=27.8+/-1.48 degrees Celsius
- Air flow rate: 28.3 litres per minute
- Air change rate: Not reported
- Method of particle size determination: Not determined
- Treatment of exhaust air: Not reported


TEST ATMOSPHERE
- Brief description of analytical method used: Scott Model 216 Hydrocarbon Analyzer
- Samples taken from breathing zone: no


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were monitored hourly using a Scott Model 216 Hydrocarbon Analyzer calibrated with known concentrations of fuel oil. Methane was used as an internal standard.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: Not reported
- Length of cohabitation: Not reported
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
11 days (gestational days 6 through 15)
Frequency of treatment:
6 hours a day
Duration of test:
14 days
No. of animals per sex per dose:
Twenty pregnant dams per dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Stated as selected by API.
- Rationale for animal assignment (if not random): Assigned sequentially to the dose groups
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations included general appearance, behaviour and condition.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: On gestational days 0, 6, 15, and 20


FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No



WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Visceral and thoracic organs


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- 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: one-third per litter
- Skeletal examinations: Yes: two-thirds per litter
- Head examinations: Yes: one-third per litter
Statistics:
Continuous parameters were analyzed with Dunnet's t-test. A 2x2 contingency table with Yates correction was used for ratio data. Discontinuous data (e.g., number of abnormal foetuses per litter) were analyzed using Wilcoxon Rank Sum test.
Indices:
Nidation index, liver and dead foetuses, implantations, pup weight, sex ratio, and abnormalities
Historical control data:
Historical data on 328 litters was provided for reproductive performance. The concurrent control was considered to be similar to the historical control.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no deaths or clinical observations due to treatment. There were no changes in reproductive indices.
Dose descriptor:
NOAEC
Effect level:
408.8 ppm (analytical)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
408.8 ppm (analytical)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was a slight (not statistically significant) increase in unusual skeletal variations in the high-dose group. These changes were related to bone ossification and not really malformations. Neither the frequency or character of the skeletal abnormalities were considered adverse for foetal growth or development.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
There were no statistically significant or treatment related adverse effects on reproductive or developmental toxicity in this study leading to a NOAEC of 2,150 mg/m3 (408.8 ppm), the highest dose tested.
Executive summary:

The effect of inhalation of No.2 Home Heating Oil (API 78-4; a mixture of straight run gas oil and cracked gas oil, CAS no 684676 -30 -2) vapour on foetal development was tested in three groups of pregnant female CRL; COBS CD (SD) BR rats. The 11 week old females were randomly assigned into groups of 20 animals each and treated (whole body) with measured concentrations of 0 (control group), 86.9 or 408.8 ppm (equivalent to 460 or 2,150 mg/m3) test substance from days 6 – 15 of gestation for 6 hours each day. The vapour was generated by passing compressed air through a warmed flask containing the test substance. No further details of vapour generation were provided and the size distribution of the resultant particles generated was not indicated. Concentrations of test material were monitored hourly using a hydrocarbon analyzer. Body weights were evaluated on gestation days 0, 6, 15 and 20; food consumption in grams was measured during the periods of gestation days 0-6, 7-15, and 16 -20 and general appearance, behaviour and condition were observed daily. On gestation day 20, the animals were anaesthetised and examined for changes in thoracic and visceral organs, number and placement of implantation sites, live and dead foetuses and number of resorption sites. The uterus and ovaries were preserved for possible future examination. A third of the foetuses from each litter were fixed for examination of soft tissues of the head, thoracic and visceral organs and the remainder were processed for skeletal examination.

There were no treatment related deaths or changes in appearance of dams throughout the duration of the study. One female delivered before the computed gestation day 20 and her pups were examined but not included in statistical analysis. Two of the mothers in the low-dose group had mottled lungs, one of which had dark red foci on the lungs. Mottled lungs were also seen in two control group animals and were not considered to be treatment related. Other conditions noted include a distended, fluid filled uterus in one animal; a clot in an implantation site in another; one animal with a dark red liver; dark red material in the right uterine horn of another animal and milky white fluid in both uterine horns in a single animal in the higher dose group. These effects appeared to be random and unrelated to treatment.

There were no statistically significant changes in uterine contents. Subcutaneous haematomas were observed in all groups – 7, 4 and 3 in control, low- and high-dose groups respectively. One foetus in the low-dose group was an unusually light colour and another female foetus had internal hydrocephaly. Most of the skeletal abnormalities observed (reduced ossification of parietal, intraparietal and hyoid bones) were considered by the authors to be common in animals of this strain. Three, 2 and 7 animals in the control, low- and high-dose groups, respectively had unusual skeletal variations (retarded bone ossification) that were not statistically significant and was not considered to be adverse for foetal growth and development.

There were no statistically significant or treatment related adverse effects on reproductive or developmental toxicity in this study leading to a NOAEC of 2,150 mg/m3, the highest dose tested.

This study received a Klimisch score of 2 and is classified as reliable with restrictions because it is well document and was generally conducted according to OECD 414 guidelines. No information was available on GLP compliance.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 110 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
One of two inhalation studies investigating the developmental toxicity of structurally related materials
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One of three dermal studies investigating the developmental toxicity potential of structurally related materials.
Additional information

Results are available from studies involving inhalation exposure of pregnant rats to either diesel oil or No.2 Home Heating Oil (a mixture of straight run gas oil and cracked gas oil) vapour. No treatment related effects on maternal health or foetal development were observed in either study, giving a NOAEC >2,110 mg/m3. The studies were well conducted and reported but the exposure period (GD 6-15) was less intensive than that recommended by current guidelines (GD 5-20) and only covered the period of organogenesis in the rat. The results do however indicate that inhalation exposure to gas oil vapour is unlikely to adversely impact foetal development.

In the first inhalation teratology study (API 1979), diesel fuel was administered via whole body exposure of pregnant female (CRL: COBS CD (SD) BR) rats to graded measured airborne (vapour) of 0, 101.8, or 401.5 ppm (equivalent to 0, 530, and 2110 mg/m3, respectively) on days 6 through to 15 of gestation. The pregnant female rats were approximately 11 weeks of age at the time of the first dose and were assigned sequentially to treatment groups of 20 animals each. There were no deaths during the course of the study and all females were normal in appearance. Mean body weights indicated no significant differences between control and treated pregnant rats, although the food consumption of the high-dose group was significantly lower (p < 0.05) than the controls during treatment. Diesel fuel did not appear to have any effect on the uterus and examination of the offspring revealed no visible abnormalities except for the occurrence of subcutaneous haematomas in 1/188, 4/227 and 4/188 in the control, low-dose, and high-dose groups, respectively. All litters appeared normal and the sex ratio did not differ significantly between treated and control groups. The conclusion of the authors was that exposure of rats to diesel fuel (vapour) did not result in compound-induced abnormalities, variation in sex ratio, embryo toxicity or inhibition of foetal growth and development. Therefore the NOAEC is >2,110 mg/m3 (401.5 ppm), the highest concentration tested.

In the second study, the effect of inhalation of No.2 Home Heating Oil, a mixture of straight run gas oil and cracked gas oil, ( API 1979) vapour on foetal development was tested in three groups of pregnant female CRL; COBS CD (SD) BR rats. The 11 week old females were randomly assigned into groups of 20 animals each and treated (whole body) with measured concentrations of 0 (control group), 86.9 or 408.8 ppm (equivalent to 460 or 2,150 mg/m3) test substance from days 6 to 15 of gestation for 6 hours each day. There were no treatment related deaths or changes in appearance of dams throughout the duration of the study. One female delivered before the computed gestation day 20 and her pups were examined but not included in statistical analysis. There were no statistically significant changes in uterine contents. Subcutaneous haematomas were observed in all groups – 7, 4 and 3 in control, low-dose and high-dose groups respectively. There were no statistically significant or treatment related adverse effects on reproductive or developmental toxicity in this study leading to a NOAEC of > 2,150 mg/m3, the highest dose tested.

A recent study to OECD 414 (dermal) and GLP was conducted on a member of the VHGO Category. This showed low systemic and developmental toxicity (NOAEL 600 mg/kg/day), with higher doses not possible due to excessive local irritation. This study was conducted on a substance in the category low in PAH content (the constituent expected to drive toxicological hazard).

In a developmental toxicity screening study, straight run diesel in acetone was dermally administered to 14 or 15 presumed pregnant rats/dose at dose levels of 0, 125, or 250 mg/kg bw/day from days 0 through 20 of gestation. Another group of 15 rats received 1000 mg/kg/day from days 0 to 5 of gestation ( ARCO 1994). Dermal irritation occurred in all treatment groups. Body weight, body weight gain, and food consumption was reduced in the 250- and 1000 -mg/kg/day groups. The maternal LOAEL is 125 mg/kg/day, based on dermal irritation.  There is no maternal NOAEL. Pup weight was decreased on lactation days 0 and 4 in the 250 -mg/kg/day group. There were no developmental effects observed in the 1000 -mg/kg/day for 6 days. The developmental LOAEL is 250 mg/kg/day, based on reduced pup weight. The developmental NOAEL is 125 mg/kg/day.  

In an additional supporting developmental toxicity screening study, VDF diesel in acetone was dermally administered to 14 or 19 presumed pregnant rats/dose at dose levels of 0, 125, or 250 mg/kg bw/day from days 0 through 20 of gestation. Another group of 14 received 1000 mg/kg/day from days 0 to 5 of gestation (ARCO 1994). Dermal irritation occurred in all treatment groups. Body weight, body weight gain, and food consumption was reduced in the 250- and 1000 -mg/kg/day groups. The maternal LOAEL is 125 mg/kg/day, based on dermal irritation. There is no maternal NOAEL. Pup weight was decreased on lactation day 0 in all dose groups and on lactation day 4 in the 250 -and 1000 -mg/kg/day groups. The developmental LOAEL is 125 mg/kg/day, based on reduced pup weight. There is no developmental NOAEL.

These studies were classified as reliable with restriction, because the high-doses had to be discontinued due to maternal discomfort and were not available to determine developmental toxicity. The studies were well conducted and were GLP compliant.

In a further developmental embryo-fetal toxicity and teratogenic potential study, straight run diesel in acetone was dermally administered to 25 presumed pregnant rats/dose at dose levels of 0, 50, 150, or 300 mg/kg bw/day from days 0 through 19 of gestation (ARCO 1993). Skin reactions (erythema, oedema, atonia, and desquamation) occurred in all treatment groups. In the mid- and high-dose group, vocalization occurred in 5 or 6 of the animals. There was a slight decrease in body weight gain in the 50 mg/kg/day group with significant reductions occurring in the 150- and 300-mg/kg/day groups. Feed consumption was only reduced in the high-dose group. There were no treatment-related effects on reproduction or development (number of corpora lutea, implantations, litter size, live fetuses, resorptions, fetal body weight, or sex ratio). There were no gross external, skeletal, or visceral abnormalities observed. The maternal LOAEL is 50 mg/kg/day, based on dermal effects and reduced body weight gain. There is no maternal NOAEL. The developmental NOAEL is>300 mg/kg/day, based on no effects at the highest dose tested.

Studies are available to investigate the developmental toxicity of diesel fuel/heating oil following dermal and inhalation exposure. Although effects were observed on progeny weight in some dermal studies, the effects only occurred at dose levels that also caused maternal toxicity. Based on this information alone it is not considered possible to determine if the effects were direct on the foetus or caused indirectly by effects on the pregnant dam.

 

Justification for classification or non-classification

The information available currently on reproduction toxicity parameters is insufficient to determine the impact on human fertility. No classification is considered appropriate at this time but a testing proposal is included for a fertility study to meet data requirements for this endpoint. 

Developmental studies only observed developmental effects (reduced pup weight) at doses that also caused maternal toxicity and it was not possible to determine whether the effects were direct on the foetus or caused indirectly by effects on the pregnant dam.

Classification for developmental toxicity is therefore not considered appropriate. Additional testing is now proposed by Concawe for the VHGO Category; dermal tests to be conducted in two species (rat and rabbit).