Distillates (petroleum), heavy naphthenic

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restrictions because the study was conducted according to or similar to guideline study OECD TG 414.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Distillate aromatic extract (CAS number 64742-04-7)

IUPAC Name:

Distillate aromatic extract (CAS number 64742-04-7)

Details on test material:

This substance can be considered “worst case” by comparison to unrefined / acid treated oils, in that the extract contains higher concentrations of biologically active components than the unrefined / acid treated oils.

- Name of test material (as cited in study report): 318 Isthmus Furfural Extract
- Substance type: Untreated distillate aromatic extract
- Physical state: Liquid
- Analytical purity: Not reported
- Composition of test material, percentage of components: Total non-aromatics=22.3%; Total aromatics=77.7% (37.2% <3 ring polycyclic aromatic hydrocarbon, 23% 3-5 ring polycyclic aromatic hydrocarbon, 2.3% nitrogen-polycyclic aromatic compound, and 12.8% sulphur-polycyclic aromatic compound)
- Lot/batch No.: CRU number 86187
- Expiration date of the lot/batch: 1991-04-30
- Other: Density is 0.98 g/mL
- CAS number: 64742-04-7

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age at study initiation: Approximately 9 weeks old
- Weight at study initiation: Not reported
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Two 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: 1988-09-30 To: 1988-10-20

Administration / exposure

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: Not reported
- % coverage: Not reported
- Type of wrap if used: None
- Time intervals for shavings or clippings: Once a week


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


TEST MATERIAL
- Constant volume or concentration used: no


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

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: Twelve days
- Further matings after two unsuccessful attempts: No
- Proof of pregnancy: Vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Days 0 - 19 of gestation

Frequency of treatment:

Daily

Duration of test:

Through gestation or postpartum day 4

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 8, 30, 125, 500, or 1000 mg/kg/day
Basis:
other: Concentration applied based on body weight

No. of animals per sex per dose:

10 to 15 pregnant females per dose

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: None reported
- Rationale for animal assignment (if not random): Not reported

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations were included: Appearance, ill health, behaviour, abortion and/or dystocia, excretory function, and mortality.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Every 3 to 4 days during gestation

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

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 or on postpartum day 4
- Organs examined: Reproductive organs, liver, and thymus were closely examined, but a complete necropsy was performed.

OTHER: Haematology and clinical chemistry were also performed on the dams sacrificed on gestation day 20.

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: Live and dead foetuses

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

Tukey's test or analysis of variance with an F-test followed by Tukey's multiple comparison test

Indices:

Foetal toxicity examinations included: resorption incidences, anomalous development (gross, skeletal and visceral abnormalities) and body weight.

Historical control data:

Historical control data from this laboratory as well as other laboratories were used when necessary.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal toxicity: Red vaginal discharge was observed in at least one pregnant animals in all exposed groups. Although authors mentioned this as being dose related, no statistics are provided.
 
In general animals exposed at 125 and 500 mg/kg/day consumed less food; at this dose level significant reduction in body weight gain, net body weight gain, and gravid uterine weight occurred throughout gestation. Body weight gain was also decreased at dose level of 1000 mg/kg/day. Of the haematology parameters evaluated, platelet and white blood cell counts were significantly affected in a dose-related manner. Effects on 14 of 22 analyzed serum components were noted at the 125 or/and 500 mg/kg/day dose levels. Thymus weights were significantly reduced and liver weights increased at doses in excess of 30 mg/kg/day. 
 
Reproductive effects: Implantation was not adversely affected by treatment. The number of dams with no viable offspring was increased at dose levels from 125 mg/kg/day. Litter size was significantly lower and resorptions were significantly increased compared to controls at 125 mg/kg/day and higher. In the postnatal group (10/group), three females were found to be not pregnant, five females reabsorbed their entire litters and one dam had only two pups, which she subsequently cannibalized

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At 30 mg/kg/day, although not statistically significant, a two-fold increase over controls in the number of resorptions was observed, which the authors considered as of biological relevance. Reanalysis of the data based on litter instead of individual absorption indicates that this is not a significant effect. Treatment at 125 mg/kg/day and at higher doses resulted in decreased mean foetal body weights. A statistically significant increase in the incidence of incompletely ossified skull bones in foetuses exposed in utero to 125 mg/kg/day was observed. 

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Dose-related clinical findings attributable to furfural extract included vaginal bleeding. This may have contributed to the paleness observed in some of the animals exposed at the 125 and 500 mg/kg/day dose groups.  Several animals at these two dose levels also had decreased stool.  In the 125 and 500 mg/kg/day groups there were significantly reduced body weights, as well as reduced gravid uterine weights, carcass weights and net maternal weight gain during gestation. (See table below)  

Dose group (g/kg/day)	Net wt change days 0-20 (g)	Gravid  Uterus   Weight (g)	Carcass   weight
1 (0)	69.3	74.5	325.3
2 (8)	62.9	77.8	326.1
3 (30)	57.3	61.3	317.2
4 (125)	36.4*	14.2*	298.2*
5 (500)	22*	4.5*	284*
6 (1000)	61	44.1	328

* P<0.05 

Consistent with the above findings, those animals in the 125 and 500  mg/kg/day groups also consumed less food than the corresponding controls. At necropsy a dose-related reduction in thymus weight was recorded. An increase in relative liver weight was recorded for the 125 and  500 mg/kg/day animals and absolute liver weights were increased in those animals exposed in the 1000 mg/kg/day group. No other  treatment-related effects were noted.  

Organ weights of prenatal animals at necropsy 

Group (mg/kg/day)	Thymus		Liver
	Abs.	Rel.	Abs.	Rel.
1 (0)	.246	.076	15.239	4.6762
2 (8)	.255	.0773	15.982	4.8992
3 (30)	.205	.0646	16.28	5.1284
4 (125)	.142**	.0475**	16.557	5.2005*
5 (500)	.081**	.0284**	16.798	5.9139**
6 (1000)	.114**	.0345**	17.741*	5.3999**
7 (0)	.204	.0585	14.8	4.2801
8 (125)	.185	.0611	12.881	4.2929

* P<0.05; ** P<0.01

 

Reproductive evaluations:

Effects were recorded at dose levels of 125 mg/kg/day and greater. There was greater than two-fold increase in percent resorptions in the 30  mg/kg/day group that was considered biologically significant. Reanalysis of the data based on litter instead of individual absorption indicates that this is not a significant effect.

The data are summarized in the following table  

Parameter	Dose group (mg/kg/day)
	0	8	30	125	500	1000
Females mated	15	15	15	15	15	15
% pregnant	87	87	93	87	67	87
Dams with viable foetuses	12/13	13/13	14/14	8/13	1/10	13/13
Dams with all  resorptions	1	0	0	5	9	0
Female mortality (%)	0	0	0	0	0	0
Corpora lutea (Mean)	15.5	17.1	17.3	15.4	13.7	17.2
Implantation sites (Mean)	14.5	16.1	15.7	14	15.5	16.3
Preimplantation loss (%)	10.6	5.6	8	11.2	-4.3a	5.4
Viable foetuses  litter size (mean)	13.9	14.6	11.6	2.1**	0.2**	8.8**
viable  males (%)	49	49	46	59	50	55
viable females (%)	51	51	54	41	50	45
Resorption (mean %)	11.8	9	27.3	82.3**	98.8**	44.9**
Dams with resorptions (%)	54	92	79	100*	100*	92

*P<0.05; ** P<0.01  

In the 500 mg/kg/day group embryos were apparently resorbed early in gestation such that some of the corpora lutea were regressed and could  not be counted. Consequently the number of implantation sites exceeded the number of corpora lutea. Hence, the negative value for mean preimplantation loss for this group.  

Changes in haematological parameters only occurred in the 125 and 500 mg/kg/day groups and were:  
125 mg/kg/day        57% increase in WBC  
500 mg/kg/day        31% decrease in platelets    54% increases in WBC  

Dose-related changes in serum chemistry also only occurred in the 125 and  500 mg/kg/day groups. The authors comment that with the  exception of uric acid, sodium, potassium and inorganic phosphorus, the changes all fell outside the normal range as defined by the 10th and 90th percentiles of the historical data.  The differences noted were:   

	125 mg/kg/day	500 mg/kg/day
Urea nitrogen	+38%	+38%
Aspartate aminotransferase	--	+55%
Alkaline  phosphatase	--	+124%
Cholesterol	--	+40%
Triglycerides	-71%	-85%
Total protein	+23%	+22%
Albumin	+34%	+38%
A/G ratio	--	+36%
Uric acid	-41%	--
Sodium	--	+3%
Potassium	+11%	+16%
Phosphorus	+32%	+31%
Calcium	+10%	+14%
Iron	+174%	+192%

There was a significant reduction of foetal body weights in groups exposed to 125 mg/kg/day or greater. Foetal body weights are shown in the following  table:

Dose group (mg/kg/day)	Mean Foetal weights (g)
	All viable foetuses	Male foetuses	Female foetuses
1 (0)	3.5	3.6	3.4
2 (8)	3.5	3.6	3.4
3 (30)	3.3	3.4	3.2
4 (125)	3**	3**	2.8**
5 (500)	2.9	3.2	2.5*
6 (1000)	2.7**	2.8**	2.6**

* P<0.05      ** P<0.01 

Foetal examinations:  

At gross examination, one foetus in the 125 mg/kg/day group was oedematous and five foetuses (from 4 litters) in the 1000 mg/kg/day group  exhibited various anomalies; two were oedematous and the other three exhibited various anomalies including shortened limbs, shortened and  missing digits, shortened trunk, cleft palate and kinked tail. Although the incidence of each observation was not significant, the total number of  foetuses observed in this group was greater than that in the controls.  There were no gross observations recorded in either the control,  8 or 30  mg/kg/day groups. Skeletal anomalies considered to be treatment-related were confined to the 1000 mg/kg/day group. In this group there was a significant increase in rib malformations (costal cartilage misshapen). Other malformations observed in the study appeared randomly and at a low frequency  throughout  the groups.  Some foetal visceral anomalies were observed, but were not statistically significant from the control group.  

Post partum observations:  

Three females in the furfural extract group were not pregnant, five females resorbed their entire litters and one dam had only 2 pups which she subsequently cannibalized. Since there was only one viable litter in this group, a meaningful evaluation of post partum effects cannot be undertaken.  

Applicant's summary and conclusion

Conclusions:

The NOAEL for both maternal and developmental toxicity is 30 mg/kg/day when administered dermally based on the numerous effects observed with doses of 125 mg/kg/day and greater.

Executive summary:

Justification for Read Across

DAE is produced as a by product in the refining of lubricating oil base stocks and waxes. Straight run vacuum distillates (lubricant base stocks) are extracted with solvents such as furfural, phenol, or N-methyl-2-pyrrolidone to selectively remove the undesirable polycyclic aromatic compounds, (especially 3-7 fused ring structures). DAE can be considered “worst case” by comparison to unrefined / acid treated oils, in that the extract contains higher concentrations of biologically active components than the unrefined / acid treated oils.

Heavy paraffinic distillate aromatic extract (CAS# 64742-04-7), 318 Isthmus Furfural Extract, was tested in a dermal study during gestation days 0 to 19 for developmental effects and maternal toxicity in the Sprague-Dawley rat. Nine groups of pregnant rats were divided in three groups: prenatal, postnatal and bioavailability groups. These groups are further described below. Bioavailability group procedures and results are not detailed here.

Prenatal groups: the undiluted test sample was applied without occlusion to the shaved skin of pregnant rats at doses of 8, 30, and125 mg/kg/day on gestation days 0-19 (15/group). An additional group received the same treatment at 500 mg/kg/day on gestation days 0-16. Initially, administration of the test sample to the 500 mg/kg/day group was also scheduled for gestation days 0-19, however treatment was discontinued after gestation day 16 because a high incidence of resorption was suspected (as indicated by a red vaginal discharge observed among rats in this group). Another prenatal group received the same treatment at 1000 mg/kg/day only on gestation days 10-12, an interval at which the developing foetus is sensitive to teratogenic insult. A group of sham treated rats served as control. Prenatal groups were sacrificed on gestation day 20.

 

The postnatal group was exposed under the same conditions as the prenatal group. Postnatal animals (10/group) were dosed at 0 or 125 mg/kg/day on gestation days 0-19. Postnatal groups were allowed to deliver their offspring naturally. Pups were observed on post partum day-0 for external malformations and variations and then together with their dams, sacrificed on post partum day-4.

 

End points examined in adults included clinical signs,body weight, food consumption, haematology and serum chemistry (only prenatal groups), liver and thymus weights, and uterine and net body weights. Foetal toxicity examinations included: resorption incidences, anomalous development (gross, skeletal and visceral abnormalities) and body weight.

 

Results - Prenatal Group

 

Prenatal groups were sacrificed on gestation day 20. All mothers were necropsied and grossly examined. Uterus and ovaries were excised and examined grossly. Numbers of corporea lutea per ovary of each pregnant animal were counted. Ovaries of non-pregnant animals were grossly examined and then discarded. Number and location of implantations, early and late resorptions and live and dead foetuses were recorded.

 

Maternal toxicity: Red vaginal discharge was observed in a number of pregnant animals in all exposed groups. Although authors mentioned this as being dose related, no statistics are provided.

 

In general animals exposed at 125 and 500 mg/kg/day consumed less food; at this dose level significant reduction in body weight gain, net body weight gain, and gravid uterine weight occurred throughout gestation. Body weight gain was also decreased at dose level of 1000 mg/kg/day. Of the haematology parameters evaluated, platelet and white blood cell counts were significantly affected in a dose related manner. Effects on 14 of 22 analyzed serum components were noted at the 125 or/and 500 mg/kg/day dose levels. Thymus weights were significantly reduced and liver weights increased at doses in excess of 30 mg/kg/day. 

 

Reproductive effects: Implantation was not adversely affected by treatment. The number of dams with no viable offspring was increased at dose levels from 125 mg/kg/day. Litter size was significantly lower and resorptions were significantly increased compared to controls at 125 mg/kg/day and higher.

 

Foetal toxicity and development: At 30 mg/kg/day, although not statistically significant, a twofold increase over controls in the number of resorptions was observed, which the authors considered as of biological relevance. However, reanalysis of the data using the litter as the statistical unit (rather than individual resorptions) identifies30 mg/kg bw/day as the appropriate NOAEL for developmental toxicity. Treatment at 125 mg/kg/day and at higher doses resulted in decreased mean foetal body weights. A statistically significant increase in the incidence of incompletely ossified skull bones in foetuses exposed in utero to 125 mg/kg/day was observed. 

 

When the period of exposure was restricted to gestation days 10 –12 and the dose increased to 1000 mg/kg/day, defects in costal cartilage development were significantly increased. Two of 114 foetuses evaluated were oedematous and had cleft palates. The cleft palate finding was considered by the authors to be biologically significant and evidence of a teratogenic effect, based on a very low historical control incidences at theirs and other laboratories.

 

Results - Postnatal Group

 

In the postnatal group (10/group), three females were found to be not pregnant, five females resorbed their entire litters and one dam had only two pups, which she subsequently cannibalized. The postpartum analysis of the single viable litter of this group was not meaningful.

The NOAEL for both maternal and developmental toxicity is 30 mg/kg/day when administered dermally based on the numerous effects observed with doses of 125 mg/kg/day and greater.

This study received as Klimisch score of 1 and classified as reliable without restrictions because the study was conducted according to or similar to guideline study OECD TG 414.