Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, NC
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: Males 183 - 251g Females 146 - 194g
- Fasting period before study: No
- Housing: individually in wire mesh cages, females in plastic maternity cages with nesting material following positive evidence of mating
- Diet: Certified Rodent Lab Diet 5002, PMI Nutrition Inc., ad libitum
- Water: Reverse osmosis treated on-site drinking water ad libitum
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 22.9
- Humidity (%): 31.5 - 59.1
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 27 October 2000 to 3 March 2001

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: prepared weekly by weighing appropriate amount of test article and adding sufficient vehicle

VEHICLE
- Concentration in vehicle: 0, 0.4, 2, 10, 25/17.5 mg/mL
- Amount of vehicle: 2mL/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until positive evidence of mating or up to 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no, after 14 days with no evidence of mating, female placed in plastic cage with nesting material
- After successful mating each pregnant female was caged: individually in plastic cage with nesting material
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability established before start of study.
Weekly for first 4 weeks then monthly analysis of formulations for test article concentration during the study
Duration of treatment / exposure:
Administration for minimum of 70 days prior to mating.
Males dosed throughout mating and until scheduled necropsy.
Females dosed throughout mating, gestation and until lactation day 20.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 17 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.8, 4, 20, 50/35 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: not stated

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: males weekly, females weekly until positive evidence of mating then gestation days 0, 4, 7, 11, 14 and 20 and lactation days 1, 4, 7, 14 and 21

FOOD CONSUMPTION: Yes
- Time schedule for examinations: males weekly, females weekly until positive evidence of mating then gestation days 0, 4, 7, 11, 14 and 20 and lactation days 1, 4, 7, 14 and 21
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day and g food/kg body weight/day: Yes

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


Oestrous cyclicity (parental animals):
Vaginal smears were prepared daily to determine the stage of the estrous cycle beginning 14 days prior to pairing and continuing until evidence of mating was observed. For females with no evidence of mating, smearing continued until the end of the mating period. The average length was calculated for complete estrous cycles (i.e. total number of returns to metestrous (M) or diestrous (D) from estrous (E) or proestrous (P). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in the next cycle.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
right testis weight, right epididymis weight, sperm motility (CASA) sperm morphology (light microscopy).
left testis and epididymis weighed and homogenized - evaluated for homogenization resistant spermatid count and sperm production rate (CASA)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter when possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, nipple retention (males)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following completion of mating period
- Maternal animals: All surviving animals on lactation day 21

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Males - epididymides (total and cauda), pituitary, prostate, seminal vesicles with coagulating glands and testes were weighed.
Males - coagulating glands, pituitary, prostate, seminal vesicles, testes with epididymides and vas deferens and gross lesions were preserved for possible future examination.
Females - no organs weighed
Females - gross abnormalities retained for possible future examination
Females that did not deliver or with total litter loss - if evidence of macroscopic implantation then number of implantation sites and corpora lutea recorded. If no evidence of implantation then uteri placed in 10% ammonium sulfide solution for detection of early implantation loss.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on post-natal day 21

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Gross findings retained for possible future examination.
Statistics:
All analyses were conducted using two-tailed tests except as noted.
The litter was used as the experimental unit.
Chi-squared test with Yates correction factor - prenatal mating and fertility indices
One-way ANOVA with Dunnett's test - pre-coital intervals, estrous cycle lengths, parental weekly gestational and lactational body weight and food consumption data, gestation lengths, sperm numbers, sperm production rates, organ weights, pup numbers, live litter sizes, pup body weight
Kruskal-Wallis test with Mann-Whitney U test - sperm motility, sperm morphology, pup sexes at birth (% males per litter), post-natal survival
Reproductive indices:
Male Mating index
Female Fertility index
Male Fertility index
Offspring viability indices:
Mean litter size
Mean post-natal survival

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs noted at 50/35 and 20 mg/kg/day and attributable to treatment included impaired use of hindlimbs, reduced hindlimb resistance, hunched appearance, lacrimation, red material on nose, eyes, mouth and/or limbs, abdominal, urogenital and/or preputial areas or at the base of the tail, yellow material on various body surfaces and generally unkempt appearance. Also noted were abnormal gait, hypoactivity, shallow and/or laboured respiration and exophthalmus.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
At 50/35 mg/kg/day 5 males and 11 females were found dead and 5 males and 3 females were killed due to poor clinical condition, generally during the first 4 weeks of treatment. At 20 mg/kg/day 4 males and 7 females were found dead and 1 male and four females were killed due to poor clinical condition generally between weeks 10 and 11 of treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean weekly body weight gains in males at 50/35 mg/kg/day were lower up to week 4, comparable to control thereafter until the first week of mating when losses were noted. Overall, body weight gain for 50/35 mg/kg/day males was 13.7% lower than controls over the dosing period. Reduced body weight gain was also noted for 20 mg/kg/day males during the dosing period and the overall weight gain was 5% lower than controls.

For females at 50/35 mg/kg/day mean body weight gain was lower during the first 4 weeks of treatment. Mean overall body weight gain for the gestation period was also lower for this group, the difference occurring during the last week of gestation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Weekly food consumption for 50/35 mg/kg/day males was lower during the first 2 weeks of dosing and for the first week of dosing for females in the same group.

For females during the lactation period, lower food consumption was noted in the 50/35 and 20 mg/kg/day groups.
Food efficiency:
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
Mean absolute prostate and pituitary gland weights were lower in 50/35 and 20 mg/kg/day males. In addition, mean absolute seminal vesicle, left and right epididymal and left and right testicular weights were lower in 50/35 mg/kg/day males.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no findings directly attributable to treatment in animals that died early.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Individual variation in estrous cycle was noted in all groups. Mean estrous cycle length in treated groups was comparable to control.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There was no effect of treatment.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The mean number of days between pairing and coitus was higher in the 50/35 mg/kg bw/day group compared to control and historical control data (4.9 days vs 2.7 days).

There were no effects on mating and fertility indices.

The mean length of gestation at 50/35 mg/kg bw/day was higher than control and historical control (22.7 days vs 22.0 days or 22.3 days respectively).

Two females in the 50/35 mg/kg bw/day group died during delivery. While there were no other overt signs of dystocia, an number of females that died (see above) did so at or near the time of parturition.

Mean number of implantation sites was reduced in 50/35 mg/kg bw/day females

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 50/35 mg/kg/day increased number of days between pairing and coitus and increased mean gestation length, reduced mean number implantation sites, mean live litter size and number of pups born reduced.
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mortality and clinical signs consistent with pathological changes in muscles and reductions in body weight gain and food consumption noted at 50/35 and/or 20 mg/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs of dehydrated appearance and bodies cool to touch were noted at a higher incidence at 50/35 and 30 mg/kg bw/day.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Total litter loss was noted for 4 females at 50/35 mg/kg bw/day and 1 female at 20 mg/kg bw/day. With the exception of 1 female at 50/35 mg/kg bw/day all litter losses occurred between lactation days 0 and 4. Furthermore, 2 females at 50/35 mg/kg bw/day and 1 at 20 mg/kg bw/day that were killed on gestation day 25 only had early resorptions in utero.

Mean live litter size and mean number of pups born in the 50/35 mg/kg bw/day group were lower than control and historical control data. A similar but less marked effect was also noted in the 20 mg/kg bw/day group.

The number of pups found dead or missing, presumed cannibalized, was higher in the 50/35 and 20 mg/kg bw/day groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain was lower in 50/35 and 20 mg/kg bw/day groups from postnatal day 4.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings attributable to maternal treatment with the test article.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mean post-natal survival and pup weights reduced at 50/35 and 20 mg/kg/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1 - Survival

Dose
(mg/kg/day)

Control

0.8

4

20

50/35

Males

Number in group

25

25

25

25

25

Found dead

0

0

0

4

5

Killed

0

0

0

1

5

Survived until termination

25

25

25

20

15

Females

Number in group

25

25

25

25

25

Found dead

0

0

0

7

11

Killed

0

0

0

4

3

Survived until termination

25

25

25

14

11

Table 2 - Mean Gestation length (days)

Dose
(mg/kg/day)

Control

0.8

4

20

50/35

Number of animals

20

22

23

15

15

Gestation length

22.0

21.9

21.9

22.0

22.7**

** p<0.01

Table 3 - Summary of Implantation Sites

 

Dose
(mg/kg/day)

Control

0.8

4

20

50/35

Implantation sites

Mean
SD
N

15.4
2.54
20

16.4
1.68
22

17.0
1.98
23

14.4
3.31
15

12.1**
4.96
15

Number born

Mean
SD
N

14.8
2.69
20

15.3
1.58
22

16.0
2.20
23

13.5
3.54
15

8.7**
4.92
15

Unaccounted sites

Mean
SD
N

0.7
0.81
20

1.1
1.04
22

1.0
1.19
23

0.9
0.99
15

3.5**
5.42
15

** p<0.01

Only dams that delivered 1 or more pups included in above table

Table 4 - Absolute Organ Weight (g)

 

Dose
(mg/kg/day)

Control

0.8

4

20

50/35

Prostate

Mean
SD
N

1.10
0.225
25

1.08
0.244
25

1.04
0.193
25

0.93*
0.200
20

0.86**
0.188
15

Seminal vesicles

Mean
SD
N

1.95
0.384
25

1.91
0.308
25

1.87
0.299
25

1.86
0.308
20

1.58**
0.410
15

Left Testis

Mean
SD
N

1.83
0.157
25

1.77
0.280
25

1.82
0.343
25

1.77
0.132
20

1.68
0.219
15

Right Testis

Mean
SD
N

1.85
0.204
25

1.81
0.148
25

1.76
0.178
25

1.76
0.109
20

1.70
0.207
15

Left Epididymis

Mean
SD
N

0.70
0.068
25

0.69
0.100
25

0.68
0.064
25

0.68
0.079
20

0.63
0.082
15

Right Epididymis

Mean
SD
N

0.71
0.048
25

0.73
0.070
25

0.69
0.071
25

0.67
0.070
20

0.65*
0.084
15

Left Cauda Epididymis

Mean
SD
N

0.3065
0.03231
25

0.3088
0.05556
25

0.2933
0.03829
25

0.2893
0.03786
20

0.2756
0.05086
15

Right Cauda Epididymis

Mean
SD
N

0.2972
0.02999
25

0.3073
0.03786
25

0.2820
0.03070
25

0.2758
0.02949
20

0.2733
0.03788
15

Pituitary

Mean
SD
N

0.0152
0.00291
25

0.0139
0.00202
25

0.0145
0.00279
25

0.0134*
0.00216
20

0.0119**
0.00186
15

* p<0.05 ** p<0.01

Table 5 - Litter Data

 

Dose
(mg/kg/day)

Control

0.8

4

20

50/35

Number born

Mean
SD
N

14.8
2.69
20

15.3
1.58
22

16.0
2.20
23

14.0
3.57
17

8.7**
4.92
15

Sex at birth
(% male)

Mean
SD
N

51.9
13.96
20

51.8
7.71
22

52.0
16.19
23

46.2
15.33
17

44.7
27.94
15

Live litter size
(PND 0)

Mean
SD
N

14.6
2.54
20

14.9
1.63
22

15.6
2.45
23

13.1
3.06
17

7.4**
5.08
15

** p<0.01

Table 6 - Postnatal survival (% per litter)

 

Dose
(mg/kg/day)

Control

0.8

4

20

50/35

Birth to PND 4 (pre-selection)

Mean
SD
N

96.6
4.85
20

95.4
6.23
22

93.3
9.03
23

82.3
33.95
15

56.8**
43.91
14

PND 4 (post selection) to PND 21

Mean
SD
N

100.0
0.00
20

100.0
0.00
20

99.5
2.61
23

89.1**
21.59
8

77.8**
40.37
6

** p<0.01

N = number of litters

If dam died during interval the litter was excluded from viability calculation

Applicant's summary and conclusion

Conclusions:
Following administration at 0, 0.8, 4, 20 or 50/35 mg/kg/day the parental NOAEL was considered to be 4 mg/kg/day based on the mortality, clinical signs and food and body weight effects and effects on male organ weights noted at 50/35 or 20 mg/kg/day. The NOAEL for reproductive performance was considered to be 20 mg/kg/day based on the increased number of days between pairing and coitus and lower number of implantation sites noted at 50/35 mg/kg/day. Based on the total litter losses, reduced postnatal survival and clinical signs and lower body weight gains noted at 50/35 or 20 mg/kg/day the NOAEL for neonatal toxicity was considered to be 4 mg/kg/day.