Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
- see below (principles of method)
Qualifier:
according to
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
- see below (principles of method)
Principles of method if other than guideline:
Deviations from guideline:
- Treatment of P generation for 56 days instead of 70 days
– No observation of oestrous cycle
– No documentation of sex ratio prior to culling of litter size
– No assessment of sperm parameters
– No determination of required organ weights
– No histopathology on coagulating gland
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: SD Crl:CD® VAF/Plus®
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: P-generation: 46 d
- Weight at study initiation: P-generation: Males: 150-190 g; Females: 119-144-x g
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Concentration in vehicle: 2.5, 7.5, 15.0 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
Details on mating procedure:
Duration of mating: maximally 21 d
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Duration of exposure before mating: 56 days (P), >= 113 days (F1)
Duration of exposure in general: From beginning of the study until sacrifice of P, F1, and F2-generation (through mating, gestation, and lactation of P and F1). F1 offspring was not exposed starting at weanling age but at 35 days of age to reduce gavage-induced injuries.
Frequency of treatment:
1x/d
Details on study schedule:
--
Remarks:
Doses / Concentrations:
25, 75, 150 mg/(kg bw*d)
Basis:
actual ingested
No. of animals per sex per dose:
26 of each sex per group
Control animals:
yes, concurrent vehicle
Details on study design:
--

Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were observed for mortality and overt signs of toxicity twice every day. During each study week, a detailed clinical observation of each animal was performed and the findings recorded daily. F1 parental animals were observed daily beginning at 22 days of age. Animals not surviving to scheduled euthanasia were necropsied and, where practicable, the tissues were preserved in fixative. Any rat showing signs of severe debility or toxicity were euthanized for humane reasons and to prevent loss of tissues. Following parturition for the F1 and F2 litters, all P and F1 males were evaluated for fertility, euthanized and necropsied. Any P male that failed to sire a litter was evaluated for spermatogenesis by examination of the epididymis for the presence of sperm.

BODY WEIGHT: Yes
Weekly until evidence of copulation or until euthanasia. Mated females were weighed on gestation days 0, 6, 15 and 20 and on lactation days 0, 7, 14 and 21. Body weights for non-gravid females were recorded on the above presumed gestation days but were not included in summarisation during gestation.

FOOD CONSUMPTION
Individual food consumption was recorded weekly for all animals prior to mating,
not measured during the mating periods because the animals were cohabited at that time.
Following the mating periods: Recorded weekly for males until euthanasia.
Weekly food consumption measurement was resumed after the mating period for females without evidence of copulation until either delivery or until euthanasia.
Mated females: Recorded on the corresponding body weight days during the gestation and lactation periods.
Non-gravid females: Recorded as indicated above but was not included in summarisation during gestation.

Oestrous cyclicity (parental animals):
not performed
Sperm parameters (parental animals):
Parameters examined in P-males that failed to sire a litter:
- Testis weight
- Presence of sperm in epididymis
Litter observations:
--
Postmortem examinations (parental animals):
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY (P, F1): Female: Vagina, uterus, ovaries; male: Testis, epididymis, seminal vesicle, prostate

ORGAN WEIGHTS (P,F1): Testis

HISTOPATHOLOGY on F1 not selected for mating, F2: Not performed, only gross necropsy with special attention on reproductive organs
Postmortem examinations (offspring):
--
Statistics:
--
Reproductive indices:
see Table under "Any other information on results"
Offspring viability indices:
see Table under "Any other information on results"
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed at 75 mg/(kg*d) and above in the F1 generation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dose-related decrease in body weight during the pre-mating period at all dose levels.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Dose-related decrease in body weight during the pre-mating period at all dose levels.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test-article-related microscopic lesions observed in animals that were either euthanized at study termination or died during the study period
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Decreased fertility and pregnancy indices in the F1 female parental rats at all dose levels, but not interpreted as a treatment-related effect: no dose-response relationship, also low in control group.
--
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: hypersalivation (males), pre-mating body-weight loss (males, females) at next higher dose (LOAEL 75 mg/kg bw/day)
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see table below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dose-related decrease in growth of the F1 generation starting at 25 mg/(kg*d) (see below)
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The decrease in mean absolute testis weight is considered secondary to decreases in body weight and not a direct effect of the test article.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Description (incidence and severity):
No test-article-related microscopic lesions observed in animals that were either euthanized at study termination or died during the study period
VIABILITY (OFFSPRING):
There was a significant reduction in the number of live F1 offspring in the mid (weak effect) and high dose (pronounced effect) groups.
There is no notable evidence of impairment of viability in F1-offspring (neonates of P) at 25 and 75 mg/(kg*d) (Tab. 19). No impairment of viability is notable in F2-offspring (neonates of F1) at 25 mg/kg, but at 75 mg/kg at day 0 (Report, Tab 24).

BODY WEIGHT (OFFSPRING):
There is dose-related mean body-weight reduction in the pup weight of the F1 generation at 14 to 21 d lactation (by about 10 %) at >= 25 mg/(kg*d) (Report, Tab. 20): not conclusive for the low-dose group and not considered as a pathologically relevant, adverse effect (see also high variation e.g. F1 + F2 controls).

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced body weight during lactation at next higher dose (LOAEL: 75 mg/kg bw/day)
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced pup viability at next higher dose (LOAEL: 75 mg/kg bw and day)
Reproductive effects observed:
not specified

Table for repoductive toxicity study:


Genera-tion

Control

Low dose

Medium dose

High dose

Pathology

m

f

m

f

m

f

m

f

Histopathological examination

No treatment-related findings

Reproductive Performance

Mating index

%

P

96.0

96.2

100

100

100

100

100

100

F1

91.7

92.3

83.3

85.2

60.0*

60.0
**

82.6

87.0

Fertility index
(m. fertile/m. paired)
(f. pregnant/f. paired)
(Tab. 18/23)

%

P

88.0

88.5

76.9

76.9

83.3

83.3

84.6

84.6

F1

50.0

61.5

29.2

25.9*

28.0

28.0*

47.8

47.8

Pregnancy index
(f. pregnant/f. mated)

%

P

--

92

--

76.9

--

83.3

--

84.6

(Tab. 18/23)

F1

--

66.7

--

30.4*

--

46.7

--

55

Number of implantation sites (Tab. 28 and 29)

Mean

P

14.0

13.4

13.2

12.5

F1

11.5

12.3

12.5

9.4

Duration of pregnancy
(Tab. 18 and 23)

Mean (d)

P

22.1

22.4

22.4

22.9

F1

22.4

22.3

22.8

23.4

Litter size
(Tab. 19 and 24)

Mean

F1

13.2

12.3

11.5

9.2* #

F2

9.9

11.7

10.0

3.4** #

Live offspring
(Tab. 19 and 24)

Mean

F1

13.0

12.2

11.1*

6,8**

F2

9.6

11.1

7.3*  #

1.8**  #

Live birth index (d 0)
(Tab. 19 and 24)

%

F1

98.6

99.1

96.1

73.7**

F2

96.8

95.1

73.3*

51.6**

Viability index (d 4)
(Tab. 19 and 24)

%

F1

98.5

94.4

85.4

76.9**

F2

85.6

80.8

100

87.5

Weight of live pups on day of age 0 (Tab. 20/25)

Mean (g)

F1

6.1

6.0

5.7

5.9

F2

6.2

5.8

6.0

6.2

Weight of live pups on day of age 14 (Tab. 20/25)

Mean (g)

F1

34.2

31.0**

29.0**

24.8**

F2

31.0

32.6

27.6

23.9*

Weight of live pups on day of age 21 (Tab. 20/25)

Mean (g)

F1

56.4

53.6

51.4**

49.9*

46.3**

44.7**

39.8**

39.0**

F2

51.1

48.4

53.3

51.1

46.1

44.5

39.3

38.0*

*significantly different from controls, p≤ 0.05

 

**significantly different from controls, p≤ 0.01

 

#  Note: in the report, Tab. 19 and 24, no statistical significance stated, and for 1.8 only p<0.05 given. This appears to be erroneous and is assumed to be p<0.05 and 0.01, respectively.

 

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Species:
rat
Additional information

The results were obtained from a structure-related tar oil containing a high level of BaP. Read across to this test material is considered to be a conservative approach as compared to the anthracene oil containing less than 50 ppm of BaP.

No typical adverse effect on reproductive performance of either sex was noted; the adverse effects observed appear to relate to pre- and post-natal development of the offspring rather than impairment of fertility.


Short description of key information:
In a two-generation reproduction study (OECD 416), the overall LOAEL / NOAEL are 75 and 25 mg/(kg bw*d), respectively, based on decreased body weight gain in the two highest dose groups (in F1 parents) and during gestation in F0 parents and decreased litter size in the high dose group, decreased survival and an increase in the number of stillborns, and decreased body weight of live pups in the two highest dose groups (less than 10 % bw reduction in the low dose group).

Effects on developmental toxicity

Description of key information
In a developmental/teratogenicity study (OECD 414), an increase in the incidence of post-implantation losses and a reduction in the number of live foetuses was seen at a dose of 175 mg/(kg bw*d) given orally to pregnant rats from gestation day 6 through 15. No adverse effect was demonstrated for late intrauterine development of live foetuses at any dose.
Link to relevant study records
Reference
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: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
- Soft-tissue examinations were performed on 1/3 instead of 1/2 of the foetuses. Post-treatment period of five days prior to caesarean section: Acc. to new OECD 414 (22 Jan. 2001), dosage should cover the whole pregnancy up to 1 d prior to scheduled caes
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: SD, Crl:CD® VAF/Plus®
Details on test animals 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 g
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE

- Concentration in vehicle: 2.5, 7.5, 22.5 mg/ml
- Amount of vehicle (if gavage):10 ml/(kg bw*d)
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Mating period: Until detection of copulatory plug (= day 0 of gestation)
Duration of treatment / exposure:
Day 6-15 of gestation
Frequency of treatment:
1x/d
Duration of test:
Duration of exposure: Day 6-15 of gestation
Postexposure period: Day 16-20 of gestation.
Remarks:
Doses / Concentrations:
25, 50, 175 mg/(kg bw*d)
Basis:
actual ingested
No. of animals per sex per dose:
30 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
--
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily on days 6-20

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 9, 12, 16 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, days 0, 6, 9, 12, 16 and 20
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

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


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice 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: Number and location of viable and non-viable fetuses
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No data

Litter size, number of dead foetuses, foetal weight, sex ratio, external alterations
Statistics:
--
Indices:
--
Historical control data:
--
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
175 mg/(kg*d) dosage level: weak maternal toxicity as evidenced by increased hair loss, body weight loss during the first subinterval of treatment (gestation days 6-9), inhibition in body weight gain during the treatment period (days 6-16) and throughout gestation (until day 20), and decreased food consumption. There was a decreasing trend only without clear statistical significance.
50 mg/(kg*d) dosage level or lower: No evidence of maternal toxicity.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Developmental toxicity at the 175 mg/(kg*d) dosage level: Increased incidence in post-implantation loss (including three whole litter resorptions vs. one litter resorbed in the vehicle control and none in the other treated groups), and reduced number of live foetuses. No adverse effect was demonstrated for late intrauterine development of live foetuses at any dose.
No evidence of developmental toxicity was observed at the 50 mg/(kg*d) dosage level or lower.
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.
Developmental variations that occurred did not follow a dose-related pattern.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
175 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
175 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

 Table forMaternal Effects

Parameter

Control data

25  

mg/(kg*d)

50

mg/(kg*d)

175

mg/(kg*d)

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 forDevelopmentalEffects

Litter response (Caesarean section data)

Parameter

Controldata

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 forMorphologicalEffects

Examination of the foetuses(Report, Tab. 9 + 10, p. 42/43)

Parameter

Controldata

25 mg/kg/ day

50 mg/kg/ day

175 mg/kg/ day

Dose-response
+ / -

historical

study

External malformations*[%]

0.20

0.00

0.77

2.891)

1.27

Skeletal malformations*[%]

0.93

0.00

0.81

4.351)

1.72

Visceral malformations*[%]

0.41

0.58

2.21

1.33

2.50

Fetuses with malformations [%]*

0.58

3.8

3.92)

5.5

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

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

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.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Species:
rat
Additional information

The results were obtained from a structure-related tar oil containing a high level of BaP. Read across to this test material is considered to be a worse case as compared to anthracene oil (BaP =< 50 ppm).

Justification for classification or non-classification

The results are accepted as relevant for the classification of anthracene oil (BaP =< 50 ppm). The adverse effects observed require the

classification for potential hazards

--according to Directive (EU) 67/548/EEC as follows:

  • for impairment of reproduction: Repr. Cat. 3; R 62:Possible risk of impaired fertility
  • for impairment development of progeny: Repr. Cat. 3; R63: Possible risk of harm to the unborn child

-- according to Regulation (EC) No 1272/2008 as follows:

  • for impairment of fertility, reproduction and development: Repr. 2, H361: Suspected of damaging fertility or the unborn child.