Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
US Type P1/13 based on GC/MS (Selection of Key Compounds)
- Substance type: organic
- Physical state: liquid

Distilled Coal Tar, Complex Hydrocarbon Mixture

Component CAS-No. [%][US P1/13]

Naphthalene 91-20-3 9.0
1-Methylnaphthalene 90-12-0 2.3
2-Methylnaphthalene 91-57-6 5.1
Indene 95-13-6 0.9
Fluorene 86-73-7 4.2
Acenaphthylene 208-96-8 0.3
Acenaphthene 83-32-9 6.1
Phenanthrene 85-01-8 12.2
Anthracene 120-12-7 2.2
Fluoranthene 206-44-0 6.8
Pyrene 129-00-0 6.0
Benz[a]anthracene 56-55-3 0.5
Chrysene 218-01-9 1.5
Benzo(a)pyrene 50-32-8 0.5

Total of 5 remaining EPA-PAH: ca. 1.4

(Benzo[b]fluoranthene, 205-99-2 0.8
Benzo[k]fluoranthene, 207-08-9 0.3
Indeno[1,2,3-cd]pyrene, 193-39-5 0.1
Dibenzo[a,h]anthracene, 53-70-3 not identified
Benzo[ghi]perylene, 191-24-2 <0.1

Test animals

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

Administration / exposure

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:
--
Doses / concentrations
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

Examinations

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"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

--

Effect levels (P0)

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)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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).

Effect levels (F1)

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)

Results: F2 generation

Effect levels (F2)

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)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

 

Applicant's summary and conclusion