Anthracene oil

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

as of 12 May 1981

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Creosote

EC Number:

232-287-5

EC Name:

Creosote

Cas Number:

8001-58-9

Molecular formula:

not applicable

IUPAC Name:

Creosote

Test material form:

liquid

Details on test material:

- Substance type: organic

Specific details on test material used for the study:

- Name of test material: Creosote; North American P1/P13 Creosote; North American Creosote Composite Test Material P1/P13
- Composition of test material, percentage of components: see under Test material information

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SD Crl:CD® VAF/Plus®

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: approx. 13 weeks
- Weight at study initiation: 210 – 278 grams

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubilty of test material
- Concentration in vehicle: 2.5, 5.0, 17.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations, homogeneity, and stability in the vehicle examined (Report, Vol. 1, Chap. 5.1 "Analytical Results" and Tab. 1-3)

Details on mating procedure:

Until detection of copulatory plug (= day 0 of gestation).

Duration of treatment / exposure:

day 6-15 of gestation

Frequency of treatment:

once daily

Duration of test:

Total duration: 20 days
Exposure period: day 6 - 15 of gestation
Postexposure period: day 16 - 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

175 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

30 mated females; effectively, 27, 20, 23 and 23 pregnant animals were available in the vehicle control and respective treated groups.

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily on days 6-20

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 9, 12, 16 and 20 (Report, Vol.I, Tab. 5/6, p. 36/37)

FOOD CONSUMPTION: yes (Report, Vol.I, Tab. 7, p. 38)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20: Caesarean section (Report, Vol.I, Tab. 8, p. 39-41)
- Organs examined: uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes, Report, Vol.I, Tab. 8, p. 39-41
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
- Soft tissue examinations: Yes (1/3 per litter)
- Skeletal examinations: Yes (1/3 per litter)
- Head examinations: Yes

Litter size, number of dead foetuses, foetal weight, crown-rump length, sex ratio, external alterations

Histopathological, microscopic examination for malformations was performed on 121 and 116 foetuses from 20 litters at the the top-dose level, 150 and 161 from 23 litters at the mid-dose level (Report, Vol 1, Tab. 9/10, p. 42/43). 

Statistics:

Male and female sex ratios and the proportions of litters with malformations and developmental variations were compared using the Chi-square test-criterion with Yates´ correction for 2x2 contingency tables and/or Fisher´s exact probability test to determine the significance of differences (see further details: Statistical methods summarised in Report, Vol. I, Chap. 4.7.).

Indices:

yes

Historical control data:

yes, comprehensive compilation in Report, Vol. I, Appendix D

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

increased hair loss during gestation days 6-9

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slightly depressed growth probably correlated with reduced food consumption. Overall, in the high-dose group, there was a decreasing trend only - without clear statistical significance (Report, Tab. 6, Tab. 7). No evidence of maternal toxicity was observed at the 50 mg/kg/day dosage level or lower.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Food efficiency:

not specified

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):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Increase in embryonal losses, early resorption

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Resorption of 3 whole liters / 20 dams (total) and 1 litter / 20 dams (only one viable foetus left) at the high dose level (175 mg/kg bw/d)

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

increased post-implantation loss (early resorptions) in the maternal high-dose group.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead or non-viable foetuses at Cesaeran section across all groups.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

Maternal toxic effects: yes

Systemic toxicity:
Hair loss, body weight loss, reduced food consumption at 175 mg/kg bw/day. But depression of maternal body-weight gain was not statistically significant, and there was no difference across the other groups, when corrections were made for gravid uterine weight.

Developmental toxicity (pre-natal):
At the maternal 175 mg/kg bw/day dosage level, there was clear evidence of a statistically significant increase in the incidence of post-implantation loss (including three whole-litter and one close-to-total resorptions vs. two litters resorbed in the vehicle control and none in the other treated  groups). No evidence of maternal or developmental toxicity was observed at the 50 mg/kg bw/day dosage level or lower (Report, Vol. I, Tab. in Appendix C, p. 79-86).

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

other: hair loss, reduced food consumption

2

Key result

Dose descriptor:

NOAEL

Remarks:

pre-natal development

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

early or late resorptions

pre and post implantation loss

total litter losses by resorption

Maternal abnormalities

1

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: systemic toxicity

Description (incidence and severity):

hair loss, food consumption, body weight and weight gain

2

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

placenta

uterus

Description (incidence and severity):

post-implantation loss (early resorptions)

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

See Tab. 2 under "Any other information on results ..."
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

In the maternal high-dose group, the number of total/live foetuses was decreased, yet not statistically significant (see Tab. 2 under "Any other information on results ...").
This was attributable to an increase in post-implantation loss (increase in early resorptions), statistically significant (p<0.05).

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

In the maternal high-dose group, gravid uterine weight was decreased, yet not statistically significant (see Tab. 2 under "Any other information on results ...")

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Apparent increases in the incidences of malformations were not statistically significant and are considered to have occurred by chance. 
The incidences of morphological variations were not different from those seen in the vehicle control.
See details under "Any other information on results ..."

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see details under "Any other information on results ..."

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see details under "Any other information on results ..."

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no test-article related effects

Foetotoxicity:
no effects observed

Malformations:
One, six, seven, and seven foetuses showed malformations in one, three, six, and five litters for the control, low-, mid-, and high-dose groups, respectively. The incidences for the mid-, and high-dose levels were significantly higher than for the control group. Most of the observed malformations are fairly common in rats and the values obtained for the eye malformations in this study were within historical control range. After factoring out these common eye abnormalities, each type of the remaining malformations occurred in only one or two instances per group. Thus, the malformations were not considered to be test-article related. (Report, Vol. I, Chap. 5.4).

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Remarks:

highest dose tested

Effect level:

175 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: foetotoxicity

2

Key result

Dose descriptor:

NOAEL

Remarks:

highest dose tested

Effect level:

175 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

175 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

 

Table 1: Maternal Effects
Parameter	Control data		25 mg/kg bw/day	50 mg/kg bw/day	175 mg/kg kg/day	Dose-response + / –
	historical	study
Number of dams examined	716	30	30	30	30
Pregnant females		27	20	23	23
Pregnancies [%]		90.0	66.0	76.0	76.0	–
Clinical findings during application of test substance		13	15	11	17	–
Mortality of dams [%]	0	0	0	0	0	–
Body weight gain
day 0-6		34	34	32	30	–
day 6-9		2	5	3	-2	–
day 9-12		12	12	13	12	–
day 12-16		28	21	25	21	–
day 16-20		57	65	64	51	–
day 6-16		42	38	40	31	–
day 0-20	143	133	137	136	112	–
Final dam weight change minus weight of uterus	62	64	67	66	61	–
Food consumption [g/animal/day]
day 0-6		21.2	21.0	20.2	20.5	–
day 6-9		14.6	16.7	14.4	12.9	–
day 9-12		14.1	15.5	14.1	13.6	–
day 12-16		17.9	15.0	12.6*	9.8**	+
day 16-20		25.5	28.6	29.5	30.4	–
day 6-16		15.7	15.7	13.6	11.9**	+
day 0-20		19.4	19.9	18.8	18.2	–
Necropsy findings in dams dead before end of test		No deaths occurred before end of test.
* Significantly different from controls, p ≤ 0.05 ** Significantly different from controls, p ≤ 0.01

 

Table 2: Developmental Effects Litter response (Caesarean section data)
Parameter	Control data		25 mg/kg/day	50 mg/kg/day	175 mg/kg/day	Dose- response + / -
	historical	study
Corpora lutea:mean/dam	16.8	16.6	16.0	16.3	16.0	–
Implantations:mean/dam	15.0	14.2	13.8	14.6	12.9	–
Resorptions:total/number of dams	850/708	31/27	17/20*	25/23	59/23*	+
Number of foetuses/dam		13.0	13.0	13.5	10.3	–
Pre-implantation loss [%]		11.6	13.8	10.6	12.2	–
Post-implantation loss [%]	7.3	8.1	6.2	7.4	19.9 *	+
Total number of litters		26	20	23	20	–
Foetuses / litter		13.0	13.0	13.5	10.3	–
Live foetuses / litter ratio	13.9	13.0	13.0	13.5	10.3	–
Dead foetuses / litter ratio		0	0	0	0	–
Foetus weight (mean) [g]	3.4	3.3	3.5	3.3	3.2	–
Male foetuses		3.4	3.6	3.4	3.3*^1)	+
Female foetuses		3.3	3.3	3.2	3.2	–
Uterine weight (mean) [g]	76.3	72.0	69.5	70.0	61.4	–
Crown-rump length (mean)[mm]		35	36	35	35	–
Foetal sex ratio[state ratio m/f]	0.982	1.108	0.904	1.006	1.135	–
* Significantly different from controls, p ≤ 0.05 ^1)questionable, no effect on crown length, see also female values, considered biologically irrelevant.

Table 3: Morphological Effects Examination of the foetuses (Report, Tab. 9 + 10, p. 42/43)
Parameter	Control data		25 mg/kg/ day	50 mg/kg/ day	175 mg/kg/ day	Dose-response + / -
	historical	study
External malformations^* [%]	0.20	0.00	0.77	2.89^1)	1.27	–
Skeletal malformations^* [%]	0.93	0.00	0.81	4.35^1)	1.72	–
Visceral malformations^* [%]	0.41	0.58	2.21	1.33	2.50	–
Foetuses with malformations [%]#		0.58	3.8	3.9^2) 3)	5.5^3)	+
External variations^* [%]	0.01	0.00	0.00	0.00	0.00	–
Skeletal variations^* [%]	34.4	36.7	50.4	49.7	43.1	–
Visceral variations^* [%]	1.44	10.5	11.0	7.33	1.67	–
Foetuses with variations [%]#		19.0	24.3	24.8	19.4	-

*Percentages are not given in the original study but calculated from the number of observations and the number of examined foetuses in the respective category.

# Percent foetuses with malformations or variation are calculated from total foetuses with defects from Tab.9 or 10, respectively.

¹⁾The relatively high incidences are only determined by an accumulation of 11 different skeletal and external malformations within one foetus from doe No. 56513 (Report, Appendix C, p. 100).

²⁾Because of multi-fold malformations observed in two foetuses (one with 4 and a second with 2 defects), the percentage of the number of animals with malformations is lower than the total percentage of malformations (comp. Report,Appendix C, p. 98ff). Four other foetuses carried only one structural defect as in the other dose groups.

³⁾Statistically significant increases in the number of affected litters as compared to control, no increase in defect incidence per litter (p<0.05) (Report, Vol. 1, Tab.9).

Applicant's summary and conclusion

Conclusions:

US creosote P1-13 was embryotoxic (early resorptions) with a NOAEL of 50 mg/kg bw in the presence of mild maternal toxicity (NOAEL also 50 mg/kg bw). It is unlikely that the increase in post-implantation losses are coupled to the decreased maternal food consumption, while there were virtually no other maternal toxic signs. Results indicate classification as reprotoxic cat. 2, "H361: Suspected of damaging fertility or the unborn child."

Executive summary:

The developmental toxicity of US Creosote P1/13 (0, 25, 50, and 175 mg/kg bw/day, gestational day 6 -15) was tested under GLP conditions in the rat according to OECD 414. Weak maternal toxicity and significantly increased early embryo resorption was seen at the top dose. Hence, the NOAEL was 50 mg/kg bw/day for both maternal and embryo toxicity.