Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987-12-29 to 1988-08-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because it is a well-documented (even though a few tables were missing) and acceptable study, but there was no Quality Assurance or GLP compliance statement.
Justification for type of information:
HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
Petroleum substances of the VHGO category are UVCB substances grouped within established production boundaries based on phys-chem properties and hydrocarbon type (a full justification for this grouping is given in a separate document reference); substances within the VHGO category have qualitatively similar properties. The prediction for read-across of hazard information on relevant human health endpoints in the category will be based on what is considered to be the worst-case substance.
The registered substances of the VHGO category comprise complex combinations of hydrocarbon constituents. Based on boiling point distribution, the hydrocarbons present will be predominantly in the range of C9-C30 (boiling in the range of 160-450 0C) and can be broadly characterized into distinct hydrocarbon classes, namely aliphatics and aromatics. As described in more detail in the document attached below (“VHGO PNDT Read-across Hypothesis”), there are sufficient data showing that the aliphatic constituents of petroleum products are not developmental toxicants, do not affect fertility, and do not produce reproductive organ toxicity (references to these data are included in Section 2 of the attached document). For aromatic molecules, data indicate that mainly 3-7 ring polycyclic aromatic compounds (PACs) may be associated with reproductive toxicity. These data include high boiling petroleum products with a relatively high level of PACs which do show potential for reproductive toxicity, whereas effects are absent in similar products without PACs.
It is therefore hypothesised that the reproductive toxicity of VHGO will be related to the types and levels of aromatics present, and will generally follow a pattern of increasing severity with increased percentage of 3 – 7 ring PACs. Any trend for the developmental toxicity of gas oils would thus be hypothetically described in terms of increasing aromatic content and number of fused aromatic rings. For VHGO specifically, which has predominantly 2- and 3-ring PACs, it is hypothesised that there is low potential for adverse effects in developmental reproductive toxicity tests from exposure to VHGOs.
In conclusion, there is a hypothetical case to suggest that any developmental reproductive effects observed in petroleum substances are associated with 3 – 7 ring PACs, and there is in-vitro and in-vivo toxicity data to support this hypothesis. However, there is no comprehensive investigation of fertility and the implications of interaction with the AhR receptor, which is thought to be a critical step in the underlying mechanism of action for these effects. Currently, Concawe has several research efforts ongoing to further support these hypotheses for reproductive toxicity; results will be published in peer reviewed scientific journals and will be submitted in future dossier updates as they come available.

CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
Crude oil (Petroleum, CAS 8002-05-9) is a complex combination of hydrocarbons, a so called UVCB (unknown variable composition, complex reaction products, biological materials) extracted in its natural state from the ground. It consists predominantly of aliphatic, alicyclic and aromatic hydrocarbons. It is used as a feedstock for petroleum refining operations, which separate and convert it into UVCB fractions (petroleum streams) as shown in the diagram in the attached document. Gas Oils (SRGO, OGO and VHGO) are one of these streams and are consistently distilled between the lighter stream (lower boiling pt) of Kerosine and the heavier stream (higher boiling pt) of Fuel Oil (all gas oils have an approximate carbon range of C9-C30 with SRGO at the lighter- and VHGO at the heavier end). Therefore only the hydrocarbon constituents present in crude oil that come off at this specific boiling point range will be present in VHGO with some overlap between neighbouring streams. They will share some common molecule types, which can give us some indication to the (reproductive) toxicity potential of VHGO substances when this information is available for these related streams.
It is important to note that petroleum UVCBs are not intentional mixtures of chemicals but are substances comprising of complex combinations of hydrocarbon species, produced to meet physical-chemical and technical performance specifications. Refining processes leading to these substances can vary slightly between manufacturers, which is reflected in the CAS number. For VHGO specifically, this means that the 9 VHGO CAS numbers are produced by slightly different refining processes but still leading to the same petroleum product to meet performance specifications for VHGO, and are thus expected to have similar compositional – and therefore toxicological- properties. Although the domain of this category of UVCB substances can be described by these broader parameters like boiling point / carbon number ranges corresponding to the refining processes by which the category members are produced, the characterization of the toxicity of hydrocarbon UVCBs, including VHGO, by assessing or summing the contributions of the individual constituents is not feasible because of the very large number of individual hydrocarbons and their isomers present (thousands to millions).
VHGO substances are used to manufacture distillate fuels (automotive diesel fuels, home heating oils, marine gas oils), as petrochemical intermediates or as components of formulated lubricants and additives (see Concawe identified uses of petroleum substances document: https://www.concawe.eu/wp-content/uploads/2017/05/Uses_Map_for_Website-21_December_2016.pdf). The only relevant routes of exposure for VHGO, as with most petroleum products especially those which are mainly used as fuels, are dermal and inhalation.
See Category Justification Document for more detailed information.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
animals were dosed from the beginning of conception (gestational day 0) until either gestational day 15 or 19.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: low viscosity liquid hydrocarbon
Details on test material:
- Name of test material (as cited in study report): Vacuum tower overheads; CAS number 64741-49-7
- Physical state: Liquid
- Analytical purity: Not reported
- Lot/batch No.: CRU 86270
- Expiration date of the lot/batch: 06-30-89
- Stability under test conditions: Not reported
- Storage condition of test material: Not reported
- Other: Density=0.91 g/ml

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age at study initiation: Presumed pregnant rats between 11 and 12 weeks old
- Weight at study initiation: Not reported
- Housing: Not reported, but likely individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22
- Humidity (%): 40% to 60%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light


IN-LIFE DATES: From: 1988-01-11 To: 1988-08-04

Administration / exposure

Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Dorsal area
- % coverage: Not reported
- Type of wrap if used: None
- Time intervals for shavings or clippings: On gestational day 0 and weekly thereafter


REMOVAL OF TEST SUBSTANCE
- Washing (if done): None reported


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Changed based on body weight
- Constant volume or concentration used: no



USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: One to one
- Length of cohabitation: 11 days
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: no, there is no data provided on the males in the study report
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: The study report does not indicate if female remained with the same male until pregnant or if the females were housed with different males each day.
Duration of treatment / exposure:
20 days
Frequency of treatment:
Daily
Duration of test:
24 days
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 125, 500 or 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
9 or 10 pregnant females per treatment group
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Not reported
- Rationale for animal assignment (if not random): Computer-generated random table of random numbers based on a sample size of two or seven

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once a day
- Cage side observations included: appearance, behaviour, excretory function, ill-health, mortality, abortion, pathosis, or premature delivery.


DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule for examinations: Every 3 or 4 days


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 or lactational day 4
- Organs examined: All organs were examined with special attention to the uterus and ovaries, the liver and thymus were weighed.


OTHER: Clinical chemistry parameters listed in Table 1 also were conducted in animals sacrificed on gestational day 20. Foetal spleens were weighed.
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: No
Statistics:
Prenatal and postnatal group data were processed and analyzed using the Reproduction portion of the Grosse Acquisition/Recording System. Maternal biophase and caesarean section data, and foetal data were evaluated using Fischer's exact or Dunnett's test. Foetal skeletal and visceral data were analyzed using an analysis of variance followed by Fischer's exact test. Organ weights were analyzed using Tukey's test. An analysis of variance followed by Student-Newman-Keul's multiple comparison test was used on the organ weight data.
Indices:
None provided
Historical control data:
None provided

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment-related effects noted in the dams commonly included skin irritation at the test site and decreased body weight (including decreased net weight gain, when reported), with increased resorption, decreased thymus weight and less frequent alterations in serum chemistry parameters

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Findings in the foetuses (or pups in studies where dams were permitted to litter) included decreased body weight, decreased crown-rump length, with decreased spleen size/weight and reductions in pup survival at lactational day 4.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Treatment-related moderate to severe skin irritation (including erythema and flaking) was recorded in all treatment groups, with oedema, eschar and necrosis/ulceration present at 500 and 1000 mg/kg/day. Vaginal bleeding, potentially indicative of litter resorptions, was also observed in animals from the two highest dose groups (onset and duration not known, tables missing from report).

Maternal body weight and body weight gains from dams treated with > 500 mg/kg/day for the majority of gestation were significantly lower than controls (final body weights for the 500 and 1000 mg/kg/day groups decreased by approximately. 20% and 30%, respectively, with weight gain from gestational day 0 to 20 decreased by approximately 50% and 70%, respectively). Body weight and weight gain data for high-dose prenatal dams treated on gestational day 10-12 only were largely unaffected however. Mean body weight of postnatal group females remained slightly (-6%), but significantly, decreased during the 4 day post-partum period. Net body weight change for prenatal group females treated with 500 or 1000 mg/kg/day on gestational days 0 to 19 was also significantly below those of the controls (decreased approximately 30 to 35% and 50 to 55%, respectively). Food consumption was generally decreased significantly in those groups exhibiting effects on body weight.

Absolute and relative thymus weights were decreased significantly in dams from the prenatal group receiving the test sample at 500 or 1000 mg/kg/day groups on gestational days 0 to 19 (absolute values decreased by approximately 40% and 50%; relative weights decreased by approximately 30% and 40%, respectively). No comparable effect was present however in postnatal animals on lactational day 4 following treatment with 500 mg/kg/day on gestational days 0 to 15 (relative and absolute values increased 35 to 40% relative to control). Maternal relative liver weight was increased 18 to 19% in animals from the 1000 mg/kg/day prenatal group only (absolute weight unaffected). Results of clinical chemistry analyses reported indicated that serum urea nitrogen, triglycerides, total protein, albumin, inorganic phosphorus, potassium and iron differed significantly in the 500 and 1000 mg/kg/day groups compared to the controls (independent verification not possible, tables missing from report).

The mean number of resorptions was significantly increased (9 to 14 fold) in dams treated with 500 or 1000 mg/kg/day, with a corresponding significant reduction in the number of viable foetuses (mean decreased to 6.0 or 1.6 foetuses, respectively, versus 15.8 to 16.4 in the controls). The number of dams with resorptions was also increased in these groups (3 to 4) compared to the controls (none affected). The mean number of resorptions in the 125 mg/kg/day group (1.7) was comparable to that of the control (1.0 to 1.1), however the number of viable foetuses was slightly (-13%), but non-significantly, reduced in this group. Other reproductive parameters (including number of corpora lutea, implantation sites, extent of preimplantation loss, sex ratios and number of dead foetuses) were unaffected by treatment.

Gravid uterine weight from dams treated with 500 or 1000 mg/kg/day was decreased by approximately 60% and 90%, respectively, relative to controls. This appeared related to a 14% and 22% reduction, respectively, in mean foetal weight for these groups (as well as the reduction in litter size mentioned above). Crown-rump length for foetuses from the 500 and 1000 mg/kg/day groups were also slightly (4 to 7%), but significantly decreased.

Three of 10 foetuses from 3 high-dose litters treated on gestational days 0 to 19 exhibited a variety of skeletal malformations, however no one type predominated. On a litter basis, skeletal malformations were present in 3 of the 6 litters subject to evaluation. Although the mean foetal incidence was significantly greater that that of the controls, the authors note that this was most likely due to the smaller total number of foetuses available for evaluation at this dose level. Soft tissue examination revealed a significant number of foetuses from the 500 and 1000 mg/kg/day groups with a small spleen (17% and 83% of foetuses examined, respectively, compared with zero in the controls), which was verified by spleen weight measurements (organ:body weight ratios of 0.066 and 0.034, respectively, versus 0.084 to 0.094 for the controls). Urinary tract anomalies (dilation of renal pelvis) were significantly increased in foetuses from the 125 mg/kg/day group; however, this was not dose-related.

Pup parameters on lactational days 0 to 4, including survival and body weight, were unaffected in litters from the postnatal group.

Applicant's summary and conclusion

Conclusions:
Based on the observations, maternal and foetal NOAELs of 125 mg/kg body weight/day are derived after dermal administration of vacuum tower overheads on gestational days 0 to 19 of pregnancy. 
Executive summary:

Two series of female Sprague-Dawley rats received daily dermal applications of the test sample (vacuum tower overheads) for various periods during pregnancy. The first series of animals (prenatal group) were given 0, 30, 125, 500, or 1000 mg/kg body weight/day on gestational days 0 to 19, with a sixth group treated with 1000 mg/kg/day on gestational days 10 to 12 only. This phase of the investigation included two sham control groups (one housed locally, the other in a separate room to prevent inhalation of volatile components) and all animals were sacrificed on gestational day 20. The second set of animals (postnatal group) received 0 (local control) or 500 mg/kg/day on gestational days 0 to 15 and were allowed to deliver their young prior to sacrifice on lactational day 4. The test substance was applied neat to a region of clipped dorsal skin (area and contact time not specified), the application site was left uncovered, and the animals fitted with Elizabethan collars to prevent ingestion during grooming.

Maternal observations (both prenatal and postnatal groups) included daily clinical observations and regular measurement of body weights and food consumption. Dams from the prenatal series were subjected to necropsy on gestational day 20 followed by a gross internal examination (including a detailed evaluation of the uterus and ovaries); the number and location of implantations, resorptions and foetuses was recorded. The thymus, liver and gravid uterus were weighed, and samples of thymus from the control and 500 mg/kg/day groups processed and examined microscopically (no further details; results not presented in report). Blood was analysed for a range of clinical chemistry parameters. Foetuses were weighed, examined externally and processed for visceral or skeletal evaluation (approximately one half of each litter per evaluation). The spleens of selected foetuses from both control groups and the 125, 500 and both 1000 mg/kg/day groups were weighed and organ/body weight ratios calculated.

Body weights were recorded for mothers and pups from the postnatal series on lactational days 0 and 4, prior to sacrifice (ether) and necropsy. The maternal thoracic cavity was examined grossly, the uterus excised and total number of implantation sites recorded, and thymus and liver weights recorded. Apart from gender, no further assessments were performed on the pups.

Treatment-related moderate to severe skin irritation (including erythema and flaking) was recorded in all treatment groups, with oedema, eschar and necrosis/ulceration present at 500 and 1000 mg/kg/day. Vaginal bleeding, potentially indicative of litter resorptions, was also observed in animals from the two highest dose groups (onset and duration not known, tables missing from report).

Maternal body weight and body weight gains from dams treated with>500 mg/kg/day for the majority of gestation were significantly lower than controls (final body weights for the 500 and 1000 mg/kg/day groups decreased by approximately. 20% and 30%, respectively, with weight gain from gestational day 0 to 20 decreased by approximately 50% and 70%, respectively), however body weight and weight gain data for high-dose prenatal dams treated on gestational day 10 to 12 only were largely unaffected. Mean body weight of postnatal group females remained slightly (-6%), but significantly, decreased during the 4 day post-partum period. Net body weight change for prenatal group females treated with 500 or 1000 mg/kg/day on gestational days 0 to 19 was also significantly below those of the controls (decreased approximately 30 to 35% and 50 to 55%, respectively). Food consumption was generally decreased significantly in those groups exhibiting effects on body weight.

Absolute and relative thymus weights were decreased significantly in dams from the prenatal group receiving the test sample at 500 or 1000 mg/kg/day groups on gestational days 0 to 19 (absolute values decreased by approximately 40% and 50%; relative weights decreased by approximately 30% and 40%, respectively), however no comparable effect was present in postnatal animals on lactational day 4 following treatment with 500 mg/kg/day on gestational days 0 to 15 (relative and absolute values increased 35 to 40% relative to control). Maternal relative liver weight was increased 18 to 19% in animals from the 1000 mg/kg/day prenatal group only (absolute weight unaffected). Results of clinical chemistry analyses reported indicated that serum urea nitrogen, triglycerides, total protein, albumin, inorganic phosphorus, potassium and iron differed significantly in the 500 and 1000 mg/kg/day groups compared to the controls (independent verification not possible, tables missing from report).

The mean number of resorptions was significantly increased (9 to 14 fold) in dams treated with 500 or 1000 mg/kg/day, with a corresponding significant reduction in the number of viable foetuses (mean decreased to 6.0 or 1.6 foetuses, respectively, versus 15.8 to 16.4 in the controls). The number of dams with resorptions was also increased in these groups (3 to 4) compared to the controls (none affected). The mean number of resorptions in the 125 mg/kg/day group (1.7) was comparable to that of the control (1.0 to 1.1), however the number of viable foetuses was slightly (-13%), but non-significantly, reduced in this group. Other reproductive parameters (including number of corpora lutea, implantation sites, extent of preimplantation loss, sex ratios and number of dead foetuses) were unaffected by treatment.

Gravid uterine weight from dams treated with 500 or 1000 mg/kg/day was decreased by approximately 60% and 90%, respectively, relative to controls. This appeared related to a 14% and 22% reduction, respectively, in mean foetal weight for these groups (as well as the reduction in litter size mentioned above). Crown-rump length for foetuses from the 500 and 1000 mg/kg/day groups were also slightly (4 to 7%), but significantly decreased.

Three of 10 foetuses from 3 high-dose litters treated on gestational days 0 to 19 exhibited a variety of skeletal malformations, however no one type predominated. On a litter basis, skeletal malformations were present in 3 of the 6 litters subject to evaluation. Although the mean foetal incidence was significantly greater that that of the controls, the authors note that this was most likely due to the smaller total number of foetuses available for evaluation at this dose level. Soft tissue examination revealed a significant number of foetuses from the 500 and 1000 mg/kg/day groups with a small spleen (17% and 83% of foetuses examined, respectively, compared with zero in the controls), which was verified by spleen weight measurements (organ:body weight ratios of 0.066 and 0.034, respectively, versus 0.084 to 0.094 for the controls). Urinary tract anomalies (dilation of renal pelvis) were significantly increased in foetuses from the 125 mg/kg/day group; however, this was not dose-related.

Pup parameters on lactational days 0 to 4, including survival and body weight, were unaffected in litters from the postnatal group.

Results from this investigation provide evidence of substance-related moderate or severe skin irritation in pregnant rats exposed to vacuum tower overheads at 500 or 1000 mg/kg/day during pregnancy. Maternal toxicity (as indicated by a reduction in body weight, body weight gain, net body weight gain, decreased thymus weight and alterations in serum chemistry) were apparent in these animals however it is not clear if these changes were due to systemic toxicity or secondary to marked skin irritation. Gravid uterine weights, mean foetal weights and crown rump lengths were also significantly reduced, and the number of resorptions significantly increased, in these dose groups, with foetal soft tissue examinations revealing a reduction in spleen size and weight. Based on these observations, maternal and foetal NOAELs of 125 mg/kg body weight/day are derived after dermal administration of vacuum tower overheads on gestational days 0 to 19 of pregnancy. 

This study received a Klimisch score of 2 and is classified as reliable with restrictions because it is a well-documented (even though a few tables were missing) and acceptable study, but there was no Quality Assurance or GLP compliance statement.