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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to internationally accepted guideline. No deviations from guideline protocol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test material (as cited in study report): PAC
Lot/Batch number: BUDDPAC396
Description: White crystalline powder
Purity: >98%
Stability: The stability of the test material in the vehicle was determined during the study by means of HPLC analyses.

Test animals

Species:
rat
Strain:
other: Wistar Crl:WI (WU)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation: (P) 3-5 wks
- Weight at study initiation: (P) Males: 130-135 g; Females: 67-72 g

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose 400 cps aqueous solution
Details on exposure:
Vehicle: 0.5% (w/v) methylcellulose water solution
Concentration in vehicle: 0, 2, 6, 36 mg/ml
Total volume applied: 5 ml/kg bw/day
Details on mating procedure:
2 weeks
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test material in the vehicle were determined during the study by means of HPLC analyses.
Duration of treatment / exposure:
Duration of exposure before mating: 10 weeks

Duration of exposure in general P, F1 and F2 males and females:
Parents:
- Females: From at least 10 weeks before mating, till day 21 of lactation
- Males: at least 10 weeks prior to the mating phase and throughout the mating period
F1:
The rats selected to rear the F2 generation were treated as from day 21 PN and subjected to the same dosing protocol of the Parent rats.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
10 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
180 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Male: 28 animals at 0 mg/kg bw
Male: 28 animals at 10 mg/kg bw
Male: 28 animals at 30 mg/kg bw
Male: 28 animals at 180 mg/kg bw
Female: 28 animals at 0 mg/kg bw
Female: 28 animals at 10 mg/kg bw
Female: 28 animals at 30 mg/kg bw
Female: 28 animals at 180 mg/kg bw
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Clinical signs: Yes – daily
Body weight: Yes – weekly
Food/water consumption: Yes – weekly
Oestrous cyclicity (parental animals):
Yes – daily, 3 weeks before the mating period
Sperm parameters (parental animals):
yes: testis weight, epididymis weight, enumeration of homogenisation-resistant spermatids in testes and epididymis, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology

Litter observations:
number of pups delivered (live- and stillborn)
number of live pups at day n
number of pups lost
number of litters lost entirely
number of male pups at day n
number of implantation sites
number of lost implantations
litter size
Postmortem examinations (parental animals):
Organ weights: uterus, ovaries, testes, epididymides (total and cauda), prostate, seminal vesicles and coagulating glands, brain, liver, kidneys, pleen, pituitary, thyroid, adrenal glands

Histopathology: vagina, uterus, ovaries, testis, epididymis, seminal vesicle, prostate, coagulating gland
Postmortem examinations (offspring):
Organ weights: uterus, ovaries, testes, epididymides (total and cauda), prostate, seminal vesicles and coagulating glands, brain, liver, kidneys, pleen, pituitary, thyroid, adrenal glands

Histopathology: vagina, uterus, ovaries, testis, epididymis, seminal vesicle, prostate, coagulating gland

At weaning 1 male and 1 female pup per litter was subjected to a thorough necropsy:

The following organs were preserved for possible future examination: brain, spleen, thymus, organs and tissues that showed macroscopic abnormalities

Reproductive indices:
number of females placed with males
number of males mated with females
number of successful copulations (= number of females mated)
number of males that became sire
number of pregnant females as demonstrated by the presence of implantation sites observed at necropsy.
number of females surviving delivery
number of females with liveborn and (all) stillborn pups
number of pups delivered (live- and stillborn)
number of live pups at day n
number of pups lost
number of litters lost entirely
number of male pups at day n
number of implantation sites
number of lost implantations
litter size

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
transient changes in female animals
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
transient changes in female animals
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
high dose-group: liver, kidney
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
increased number of cycles - considered to have no toxicological significance
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Parent males:
− No clinical signs or statistically significant differences in bodyweight changes, food consumption were observed.
− No changes were reported in the sperm analysis.
− No treatment-related differences were observed in the fertility and reproductive performance (pre-coital time, male fertility index)
− A statistically significant increase was recorded in relative liver weight and absolute and relative kidney weight in the high-dose group.
− No treatment related findings were reported in the macroscopic and microscopic pathology examinations.

Parent females:
−No clinical signs or statistically significant changes in food consumption were observed.
−Transient changes in the mean body weights achieving statistical significance were observed throughout the premating period in the mid- and high-dose groups.
− An increase in the number of cycles per animal was observed in the high-dose group in comparison to controls. This effect was considered as not toxicologically significant.
− No treatment-related differences were observed in the fertility and reproductive performance (mating index, female fecundity and fertility index, post-implantation loss).
− A slight but statistically significant increase in the duration of the gestation period was observed in the high-dose group in comparison to control.
−In the parental females, A statistically significant increase was recorded in relative kidney weight and absolute and relative liver weight in the high-dose group.
−No treatment related findings were reported in the macroscopic and microscopic pathology examinations of parental animals.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: increase in relative liver weight and absolute and relative kidney weight
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: increase in relative kidney weight and absolute and relative liver weight slight but statistically significant increase in the duration of the gestation period

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
but no clear dose response
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

−No effects were observed in the mortality and sex ratio of the delivered pups. A statistically significantly increased incidence in the number of runts was observed in the low- and mid-dose group throughout the weaning period, but was judged as a non treatment related effect, due to the lack of dose-response relationship. No differences were noted in the pup weights among the controls and the treatment groups.
−No macroscopic abnormalities related to treatment were observed in the stillborn and died pups and in the pups not selected to rear the F1 generation.
−No differences were recorded in the organ weights of pups among the treatment groups and the controls.

F1 males
− A male rat of the high-dose group was found dead on day 30 of the premating period. The death was not attributed to the treatment. No clinical signs were observed.
− Mean body weights of the male animals of the low- and mid-dose group were statistically significantly decreased when compared to the control group during the premating period of the F1-generation. In the high-dose group only a statistically significant decrease in body weight was observed on day 21.
−Decreased food consumption was observed in the low- and mid-dose groups.
−No effects were observed in the sexual maturation of the F1 males
−No changes were reported in the sperm analysis.
−No treatment-related differences were observed in the fertility and reproductive performance (pre-coital time, male fertility index).
− A statistically significant increase in absolute and relative kidney weight and in the relative kidney weight was recorded in F1 parental males of the high- and mid-dose group, respectively. A statistically significant increase in the relative brain weight was observed in the mid-dose group. Due to the concurrent increase in the mean bodyweight, the effects observed in the mid-dose group were considered as not treatment related.
− No treatment related findings were reported in the macroscopic and microscopic pathology examinations of the parental females.

F1 females
− 2 females (one from the low-dose group and on from the high dose group) were found dead during the premating period. A further female of the mid-dose group was sacrificed for humane reasons on day 11 of lactation. None of the deaths was attributable to the treatment.
− No clinical signs or significant changes in body weights or food consumption were observed.
− No differences were observed in the estrus cycle.
− No treatment-related differences were observed in the fertility and reproductive performance (mating index, female fecundity and fertility index).
− A slight but statistically significant increase in the duration of the gestation period was observed in the mid- and high-dose groups in comparison to control. However, due to the very low precoital time in the control group, the increase in the mid-dose group was not considered as treatment-related.
− The following statistically significant changes were observed in the high-dose group in comparison to controls: increased incidence of post-implantation loss, decrease in the number of delivered pups and increase in the incidence of stillborn pups.
− The number of runts was statistically significantly decreased in all the treatment groups in comparison to controls. An increased number of cold pups was observed in the high-dose group in comparison to controls. Both effects were considered as non treatment-related.
− Pup weights were statistically significantly increased in the high-dose group in comparison to controls during the lactation period. This finding was probably related to the lower number of pups delivered in the high-dose group. A decreased relative brain weight, attributable to the increase in bodyweight, was observed in the male pups of the high-dose group in comparison to control. No other differences were noted in the pup organ weights among the controls and the treatment groups.
− No macroscopic abnormalities related to treatment were observed in the stillborn and died pups and in the pups not selected to rear the F2 generation.
− A statistically significant increase was recorded in absolute and relative kidney weight in the high-dose group.
− No treatment related findings were reported in the macroscopic and microscopic pathology examinations.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: increased relative and absolute kidney weight increased alkaline phosphatase levers and decreased phospholipids and cholesterol levels
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: increased absolute and relative kidney weight prolonged gestation period increased incidence of post-implantation losses and stillborn pups decreased number of delivered pups

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Clinical chemistry:

The following statistically significant changes were observed in the high-dose group:

-     increased alkaline phosphatase levels in males only

-     decreased phospholipids and cholesterol levels in males only.

A statistically significant decrease in the creatinine levels was observed in females of the low-dose group and was considered as incidental and not related to treatment. Reproductive toxicity test results 2 -generation study

Parameter

 

control

low dose

medium dose

high dose

Genera­tion

m

f

m

f

m

f

m

f

Organ weights

% of control

 

 

 

 

 

 

 

 

 

Liver

Absolute

P

-

-

0

+1

0

0

+4.6

+6.6*

 

F1

-

-

-8.7

-3.1

-9.6*

-3.0

-0.6

+3.2

Liver

Relative to bw

P

-

-

0

+2.8

-1.0

+1.6

+5.6*

+6.9*

 

F1

-

-

-2.9

-0.3

-0.5

-1.9

+4.4

+3.6

Kidney

Absolute

P

-

-

+1.6

-1.0

+4.8

+1.7

+10.0**

+4.9

 

F1

-

-

-5.3

0

-1.9

+0.9

+10.6**

+4.9*

Kidney

Relative to bw

P

-

-

+2.0

0

+3.9

+3.6

+12.7**

+5.2*

 

F1

-

-

+3.6

+3.1

+8.4*

+2.2

+16.3**

+5.3*

Clinical chemistry

 

F1

-

-

 

 

 

 

 

 

Alkaline phosphatase

U/l

F1

187

398

177

383

189

387

242*

484

Cholesterol

mmol/l

F1

2.33

2.69

2.17

2.58

2.17

2.82

1.83**

2.48

Phospholipids

mmol/l

F1

2.15

2.44

1.98

2.34

1.94

2.49

1.71**

2.29

Reproductive Performance

 

Post implantation loss

Mean

P

11.07

8.31

9.49

14.89

F1

5.66

7.07

11.37

19.77**

Duration of pregnancy

Mean

P

21.52

21.46

21.48

21.91*

 

F1

21.19

21.32

21.50*

21.88**

Stillborn/pups delivered

 

P

0/258

3/300

0/265

5/247

 

 

F1

1/284

2/267

0/250

17/241**

Live birth index

 

P

100

99

100

98

 

 

F1

100

99

100

93

* = p<0.05; ** = p<0.001

Applicant's summary and conclusion

Conclusions:
Due to the effects observed, 30 mg/kg bw/day was considered as the NOAEL of the study.
Parent males: Increased relative liver and absolute and relative kidney weights at 180 mg/kg bw/day
Parent females: Increased absolute and relative liver and relative kidney weights at 180 mg/kg bw/day. Prolonged gestation period at 180 mg/kg bw/day.
F1 males: Increased absolute and relative kidney weight was recorded in F1 parental males at 180 mg/kg bw/day. A statistically significant increase in alkaline phosphatase levels and a statistically significant decrease in phospholipids and cholesterol levels were observed at 180 mg/kg bw/day.
F1 females: A statistically significant increase was recorded in absolute and relative kidney weight at 180 mg/kg bw/day. Prolonged gestation period at 180 mg/kg bw/day. Statistically significantly increased incidence of post-implantation losses and stillborn pups, statistically significantly decreased number of delivered pups at 180 mg/kg bw/day.
Executive summary:

28 rats/sex/group were exposed to 0, 10, 30, 180 mg/kg bw/day PAC by gavage. Parental animals were dosed as from 70 days before the mating period through all the mating period. Females were allowed to deliver and were treated throughout the gestation period up to day 21 of lactation.

On PN 21, the litters were weaned and 28 males and 28 females were selected at random from as many litters as possible in each group to rear the next generation.

Bodyweight changes, food intake and reproductive performance were studied in the parental and F1 generations. Furthermore, 10 rats/sex/group were subjected to full clinical chemistry examinations at the necropsy.

Post-natal survival, bodyweight and post-weaning sexual development were studied in generation F1 and F2. Any finding detected in the autopsy of the parental generation and external abnormalities found in pups were reported.

Three rats of the F1-generation (1 from the low-dose group and 2 from the high-dose group) were found dead during the premating period. In addition, one F1-female rat of the mid-dose group had to be killed on day 11 of lactation. The deaths were judged as not related to treatment. No treatment related clinical signs were observed. No treatment-related effects on body weight and food consumption were observed in P and F1 rats. Estrus cycle and cycle length, sperm analysis and other male and female fertility parameters were considered normal in both generations, with the exclusion of a statistically significant increase in the incidence of post-implantation losses observed in the F1 high-dose group. The mean duration of gestation was increased in both generations in the high-dose group.

No effect was observed on pup mortality in the F0-generation and the mean number of F1-pups delivered was comparable among the treatment groups and the control group. The total number of F2-pups delivered was statistically significantly decreased in the high-dose group when compared to the control group. The total number of stillborn pups was statistically significantly increased in the high-dose group. No treatment-related clinical abnormalities and effects on pup weight and organ weights were observed in the F1- and F2-pups.

A statistically significantly increased kidney weight (relative and or absolute) was observed in the male and female F0- and F1-rats of the high dose-group. A statistically significantly increased liver weight (relative and or absolute) was observed only in the male and female F0-animals.

Alkaline phosphatase was statistically significantly increased in the male F1-animals of the high-dose group. Cholesterol and phospholipids were statistically significantly decreased in the male F1-animals of the high-dose group. These effects were considered to be treatment-related.

At necropsy no treatment-related changes were observed at the macroscopic and microscopic pathology examinations of the P and F1 parental rats.