Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 January 1990 to 04 January 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study compliant with test guidelines of the time. However, current guidelines for two-generation reproduction toxicity studies require additional endpoints. Available as unpublished report, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
1983 (current guideline adopted 2001)
Deviations:
yes
Remarks:
(does not meet current guideline specification)
Qualifier:
according to
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Version / remarks:
1982 (current guideline adopted 1998)
Deviations:
yes
Remarks:
(does not meet current guideline specification)
Qualifier:
according to
Guideline:
other: MAFF Japan, Testing Guidelines for Toxicity Studies, No. 4200 (1985)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Canada Health Protection Branch: Guidelines for Pesticide Toxicology Data Requirements (1981)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague Dawley Crl:CD(SD)BR
- Age at study initiation: P generation approximately 6 weeks old
- Weight at study initiation: P generation males: 174-232 g; females: 143-175 g
- Housing: Individual in solid floor macrolone cages with stainless steel lids except during mating (one male with one female) and during lactation (one female with litter). Autoclaved sawdust bedding.
- Diet: Ssniff R10 powdered diet ad libitum
- Water: Tap water in plastic bottles ad libitum
- Acclimation period: P generation 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 30-70%
- Air changes (per hour): Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 18 January 1990 To: 04 January 1991

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
DIET PREPARATION
- Frequency of preparation of diet: Monthly
- Mixing appropriate amounts with diet: The particle size of the test substance was reduced and standardised prior to use using a centrifugal laboratory mill. It was then admixed to the powdered diet in several steps (e.g. 1000 g and 10 kg pre-mixtures and 50 kg final mixture)
- Storage temperature of food: room temperature control and 50, 500 and 1000 ppm dose levels, -20°C for 10 ppm level
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Maximum of 21 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from each batch of test diet prepared, together with a reference sample of control diet, were analysed for achieved concentration and homogeneity. The stability of dietary preparations had already been confirmed in earlier studies.

After overcoming initial homogeneity problems, the dietary preparations were determined to be homogeneous and close to nominal concentrations and stable for the period of use.
Duration of treatment / exposure:
P animals: For 14 weeks premating, up to 21 days mating, during pregnancy until weaning
F1 animals: For 14 weeks after weaning/premating, up to 21 days mating, during pregnancy until weaning
Frequency of treatment:
Continuous
Details on study schedule:
- Pre-mating period P Generation: 14 weeks
- Selection of F parents from F1 generation at 21 days of age
- F1 parental animals not mated until 14 weeks after weaning


Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 50, 500 and 1000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: On the basis of results from a dose-ranging reproduction study in the rat (Ciba-Geigy study number 89 1187, HLD project number 380-153).
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Parental animals once weekly during pre-mating and mating periods and prior to terminal kill. Females were additionally weighed on days 0, 7, 14 and 20 post coitum and on days 1, 4, 7, 14 and 21 post partum.

FOOD CONSUMPTION:
- Time schedule for examinations: Parental animals twice per week during pre-mating period. Additionally food consumption for females was determined for days 0-3, 3-7, 7-10, 10-14, 14-20 post coitum and days 1-4, 4-7, 7-10, 10-12, 12-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20 and 20-21 post partum (calculated as mean daily food consumption/measurement period).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OTHER:
- Date of mating
- Date of parturition
- Duration of gestation
- Abnormalities of nesting or nursing behaviour
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes to 8 pups/litter (4/sex where possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
- F1 and F2 offspring: number and sex of pups, live births, postnatal mortality, clinical condition, body weight, pinna unfolding, tooth eruption, eye opening, pupillary reflex and auditory response

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and visceral abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Males: After the last litter of each generation was weaned.
- Females: After the lactation period or approximately day 26 post-coitum.

GROSS NECROPSY: Yes
- The uteri of apparently non-pregnant females were immersed in 10% ammonium sulphide to reveal evidence of implantations.
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

ORGAN WEIGHTS: Yes (all parental animals)
- organs weighed: epididymides, kidneys, liver, testes

HISTOPATHOLOGY: Yes
- Tissues examined (control and high dose animals only): Bile duct, cervix, coagulating gland, epididymides, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, stomach, testes, uterus, vagina, any macroscopically abnormal tissue
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.

GROSS NECROPSY
- External and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS: No
Statistics:
Statistical analyses used the SAS software package release 6.03.
Analysis of variance with one factor treatment followed by the Student-Newman-Keuls test for multiple group comparisons: body weight, body weight gain, litter weight, organ weights, food consumption.
Analysis of variance with one factor treatment based on taking the ranks of the variables and followed by the Student-Newman-Keuls test for multiple group comparisons: mating performance, duration of gestation, mean pup weight, pup number, live birth index, viability indices, weaning index, pinna unfolding, hair growth, incisor eruption, eye opening.
Reproductive indices:
For both generations the parental reproductive performance was assessed and the following calculated: mating performance, insemination index, fecundity index, fertility index, gestation index
Offspring viability indices:
Live birth index, pup viability index, weaning index and sex ratio.

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):
Tables 1 & 2
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Tables 1 & 2
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: Table 3

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

BODY WEIGHT (PARENTAL ANIMALS):
P generation: Body weights of males and females reduced in a dose dependent manner at dose levels of 500 ppm and 1000 ppm during the premating phase. Body weights of females during gestation and lactation was generally similar in all groups.
F1 generation: Decreased weaning weights on day 21 post-partum resulted in lower initial F1 parental body weights at 500 and 1000 ppm. Thereafter during the premating, gestation and lactation periods body weights at 500 and 1000 ppm were dose-dependently decreased.

FOOD CONSUMPTION (PARENTAL ANIMALS):
P generation: There was a marginal reduction in food consumption for males at 1000 ppm during the premating phase. For females in all groups, food consumption was similar during the premating, gestation and lactation periods.
F1 generation: Food consumption was slightly reduced at 1000 ppm for males and females during the premating phase and for females during gestation and lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
Dietary concentrations of 10, 50, 500 and 1000 ppm are calculated to be equivalent to 0.8, 4, 40 and 80 mg/kg bw/day respectively based on food intake and body weight during the pre-mating periods.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested equivalent to 80 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
toxicity
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: equivalent to 4 mg/kg bw/day. Reduced parental body weight and food consumption, reduced pup weight at weaning, at higher doses.

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):
Table 4
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

BODY WEIGHT (OFFSPRING):
F1a: Mean pup weights at 1000 ppm were significantly reduced on day 14 and mean pup weights at 500 and 1000 ppm were reduced on day 21.
F2a: Mean pup weights at 1000 ppm were significantly reduced on day 21.

PHYSICAL & FUNCTIONAL DEVELOPMENT (OFFSPRING):
There were no treatment-related effects on the physical development (incisor eruption, pinna unfolding, eye opening) of the F1a of F2a pups or on the outcome of the functional tests (pupillary reflex, auditory response) on either generation.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Generation:
F1
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested equivalent to 80 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
toxicity
Generation:
F1
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: equivalent to 4 mg/kg bw/day. Reduced parental body weight and food consumption, reduced pup weight at weaning, at higher doses.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Intergroup comparison of mean body weight change (g) prior to and during mating

Dietary concentration of CGA154281 (ppm)

Males

Females

0

10

50

500

1000

0

10

50

500

1000

P generation

Days 1-99

307.9

292.5

298.3

277.7*

268.0**

115.2

109.5

108.6

104.2*

94.7**

F1 generation

Days 1-99

401.6

404.2

399.8

369.5

352.9**

193.6

190.1

182.5

179.5

168.8**

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

 

Table 2: Intergroup comparison of female body weight change (g) during pregnancy and lactation

 

Body Weight Change

Dietary concentration of CGA154281 (ppm)

P Generation

0

10

50

500

1000

Gestation days 0-20

106.20

111.47

104.51

108.29

108.88

Lactation days 1-21

24.69

30.59

19.70

25.77

40.07*

F1 Generation

Gestation days 0-20

117.01

114.10

110.48

105.10*

99.63**

Lactation days 1-21

28.41

42.93**

30.73

37.00

40.88*

P<0.05 * difference from control statistically significant

Table 3: Overall mean dose received (mg/kg/day)

Generation

Sex

Dietary concentration of CGA154281 (ppm)

10

50

500

1000

P Parents

Males

0.66

3.44

33.7

66.6

Females

0.79

3.99

39.9

79.7

Mean

0.73

3.72

36.8

73.2

F1 Parents

Males

0.80

4.07

44.0

86.4

Females

0.89

4.42

47.6

90.5

Mean

0.85

4.25

45.8

88.5

P + F1 Parents

Mean

0.79

3.99

41.3

80.1

 

Table 4: Pup body weights (g)

 

Pup body weight (g)

Dietary concentration of CGA154281 (ppm)

F1a

0

10

50

500

1000

day 1 post partum

6.3

6.2

6.2

6.1

6.0

day 4 post partum

8.2

7.9

8.2

7.7

7.5

day 7 post partum

11.9

11.3

11.7

10.7

10.7

day 14 post partum

24.4

23.5

23.3

22.3

21.8*

day 21 post partum

40.9

39.6

38.4

36.3*

35.8*

F2a

day 1 post partum

6.2

5.8

6.0

5.8

5.8

day 4 post partum

7.9

7.6

8.3

7.3

7.6

day 7 post partum

11.8

11.7

12.5

11.1

11.6

day 14 post partum

25.2

24.7

25.5

23.4

23.0

day 21 post partum

42.4

41.2

41.8

40.1

37.4*

P<0.05 * difference from control statistically significant by analysis of variance followed by Student-Newman-Keuls test

Applicant's summary and conclusion

Conclusions:
Dietary administration of 500 or 1000 ppm CGA154281 over two generations resulted in lower body weights of the adult animals during the pre-mating period and lower body weights of the pups at weaning. There was no effect of CGA1545281 on fertility or reproductive performance in either generation and no effect on the number and viability of the pups born.
The NOAEL for toxicity was 50 ppm, corresponding to a mean daily intake of 4 mg/kg bw/day. The NOAEL for fertility and general reproductive performance was 1000 ppm corresponding to a mean daily intake of 80 mg/kg bw/day.
Executive summary:
Groups of 25 male and 25 female Sprague-Dawley rats were given CGA154281 in the diet at nominal concentrations of 0, 10, 50, 500, and 1000 ppm continuously over two generations from the start of treatment until necropsy. After a pre-mating period, the P parental animals were mated and allowed to litter and to rear the F1a pups to weaning. After a 14 week post weaning maturation period the selected F1 parental animals were mated, allowed to litter and to rear the F2a pups to weaning. At 500 and 1000 ppm, effects in the P and Fl parental animals included reduced body weight and food consumption. For pups, mean body weight was significantly reduced on day 21 for both F1a and F2a pups at 1000 ppm and for F1a pups at 500 ppm. There were no adverse effects of 10 or 50 ppm CGA154281. There was no effect on fertility or general reproductive performance in either of the two generations at any dietary concentration of CGA154281 in this study.