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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
Study period:
26 September 1989 - 5 October 1990
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 Available as unpublished report, fully adequate for assessment.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)
Principles of method if other than guideline:
Not applicable
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): CGA185072 technical
- Analytical purity: 91.6 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Spraque-Dawley rats, strain Crl:CD (SD)BR
- Age at study initiation: P generation - approx. 6 weeks old
- Weight at study initiation: P generation - males 162-226 g, females 134-181 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: powdered Ssniff R 10 ad libitum
- Water: tap water in plastic bottles ad libitum
- Acclimation period: P generation 18 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 30-70%
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
DIET PREPARATION
- The test substance was milled to a standardised particle size and admixed to the powdered diet. Fresh preparations were made monthly


Details on mating procedure:
One male was housed with one female from the same group for up to three weeks. After 2 weeks, females with no evidence of mating were paired with a proven male from the same group. Mating was confirmed by the presence of sperm in a vaginal smear or a vaginal plug and designated day 0 of gestation. After successful mating, females and males were caged individually. Sibling pairing was avoided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All batches of test diet made were analysed for achieved concentration and homogeneity. Stability was confirmed for the period of use and storage conditions. Satisfactory results were obtained. Analysis was by HPLC.
Duration of treatment / exposure:
From the start of the P generation through to necropsy of individual animals - F1 and F2a.
Frequency of treatment:
Continuous
Details on study schedule:
- Pre-mating period P Generation: 100 days
- Selection of F parents from F1 generation at 21 days of age
- F1 parental animals not mated until 100 days after weaning
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 500, 5000 and 10000 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 basis of preliminary study
- Rationale for animal assignment: P generation by stratified randomisation based on body weight and the use randomisation tables; F1 generation selected by stratified use of randomly drawn cards
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: once weekly for males and females during the pre-mating and mating periods; for females on days 0, 7, 14 and 20 post coitum and days 1, 4, 7, 14 and 21 post partum

FOOD CONSUMPTION:
- Time schedule for examinations: twice weekly for males and females during the pre-mating period; for females on days 0-3, 3-7, 7-10, 10-14, 14-20 post coitum and days 1-4, 4-7, 7-10, 10-12, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21 post partum. Food consumption was calculated as mean daily food consumption per measuring 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 recorded
Sperm parameters (parental animals):
Not recorded
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes to 8 pups (4 males and 4 females where possible)

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):
Parental males were necropsied after mating, females after weaning. Successfully mated females which failed to produce a litter were necropsied on day 26 post coitum. All parental animals were examined for gross pathological changes. Uteri were immersed in 10% ammonium sulfide to reveal evidence of implantation. The number of implantations was recorded.
The following tissues were sampled and fixed from all F0 and F1 adult animals: bladder, cervix, coagulating gland, epididymides, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, testes, uterus, vagina and macroscopically altered organs. Histopathology was conducted in controls and top dose group animals. Epididymides, kidneys, liver and testes were weighed before fixation. In addition, the kidneys of group 4 animals were examined histologically.
Postmortem examinations (offspring):
Non-selected pups from F1 and F2 litters were necropsied after weaning.
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:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: Table 1

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality: No effects in P animals; four F1 males given 10000 ppm and found dead were considered treatment-related
Clinical signs: Five P females with fur staining considered treatment-related but finding not seen in F1 generation

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight: The males and females of the P and F1 generations given 10000 ppm had slightly lower body weights during the premating period.
Food consumption: No effect in P generation; slightly lower food consumption in F1 generation males and females.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
Dietary concentrations of 50, 500, 5000 and 10000 ppm are calculated to be equivalent to 4, 40, 420 and 830 mg/kg bw/day respectively based on food intake and body weight during the pre-mating periods.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related findings in P generation males or females. Changes in the kidneys and urinary bladder in particular were considered to be related to treatment with 10000ppm in F1 males and females. Groups given 50, 500 or 5000 ppm were unaffected.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related findings in P generation males or females. Changes in the kidneys and urinary bladder were considered to be related to treatment with 10000ppm in F1 males and females. There were a variety of inflammatory changes mainly moderate to marked subacute to chronic interstitial pyelonephritis associated with changes such as hyaline casts, tubular and pelvis dilatation associated with parenchymal atrophy, pelvis lithiasis and signs of hyperplastic response such as proliferation and metaplasia of the urothelium. Groups given 50, 500 or 5000 ppm were unaffected.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested equivalent to 830 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
toxicity
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Equivalent to 420 mg/kg bw/day. 10000ppm: reduced body weight, renal pelvis dilatation in pups.

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 2
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Table 3
Histopathological findings:
not examined

Details on results (F1)

BODY WEIGHT (OFFSPRING)
The mean pup weight of the F1a and F2a pups was significantly reduced at weaning on day 21 post partum.

PHYSICAL 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.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination revealed a high incidence of changes in the kidneys, particularly dilatation of the renal pelvis, in the F1a pups; groups treated at 50, 500 and 5000 ppm remained unaffected. A low incidence of F2a pups had dilatation of the renal pelvis not clearly treatment-related.


Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
fertility and general reproductive performance
Generation:
F1
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested equivalent to 830 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
toxicity
Generation:
F1
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Equivalent to 420 mg/kg bw/day. 10000 ppm: reduced body weight and histological changes in the kidneys of adults.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1 Test Substance Intake During The Pre-Mating Period (corrected means – mg/kg/day)

 

Males

Dietary concentration (ppm)

Females

Dietary concentration (ppm)

P Generation

50

500

5000

10000

P Generation

50

500

5000

10000

Week 1

5.2

51.2

555.4

1066.9

Week 1

5.1

49.7

548.8

1086.0

Week 14

2.8

28.0

277.9

565.9

Week 14

3.7

35.9

369.9

732.4

Mean intake

3.5

35.3

370.7

721.6

Mean intake

4.2

40.7

422.8

846.9

Overall mean intake for males and females

3.9

38.0

396.8

784.3

F1 Generation

F1 Generation

Week 1

6.2

66.6

627.1

1212.1

Week 1

6.6

69.1

673.0

1274.9

Week 14

2.9

30.7

300.9

613.3

Week 14

3.7

38.4

372.2

735.4

Mean intake

4.0

41.1

408.7

819.5

Mean intake

4.6

48.3

487.3

945.6

Overall mean intake for males and females

4.3

44.7

448.0

882.6

Overall mean intake for males and females

P + F1 generations

4.1

41

422

833

 

Table 2 Group Mean Pup Body Weight (g)

 

F1a pup

Weight (g)

Dietary concentration (ppm)

F2a pup

Weight (g)

Dietary concentration (ppm)

0

50

500

5000

10000

0

50

500

5000

10000

Day 1

6.1

5.9

6.1

6.1

6.2

Day 1

6.2

6.0

5.9

6.1

6.3

Day 4

7.5

7.1

7.3

7.6

7.6

Day 4

8.0

7.6

7.3

7.6

7.9

Day 7

11.4

10.3

10.8

11.4

10.9

Day 7

11.6

11.4

10.3

10.6

11.2

Day 14

24.5

21.6

22.9

25.2

21.8

Day 14

24.3

24.6

22.9

24.7

23.8

Day 21

41.0

36.6

38.3

39.6

34.9*

Day 21

41.8

41.0

40.3

40.0

37.7*

 P<0.05 * statistically significant difference from control

Table 3 Incidence of pups with renal pelvic dilatation

 

F1a pups

Dietary concentration (ppm)

F2a pups

Dietary concentration (ppm)

0

50

500

5000

10000

0

50

500

5000

10000

No. Examined

228

253

232

237

206

No. Examined

197

217

185

231

213

Unilateral

1

0

2

0

10

Unilateral

1

1

1

2

1

Bilateral

1

1

3

0

20

Bilateral

0

0

1

3

2

 

Applicant's summary and conclusion

Conclusions:
Dietary administration of 10000 ppm CGA185072 over two generations resulted in lower body weights of the adult animals and the pups from weaning together with microscopic renal and urinary bladder alterations in adults and pelvic dilatation in pups (essentially F1a). There was no effect of CGA185072 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 5000 ppm, corresponding to a mean daily intake of 350 mg/kg bw/day.
Executive summary:

Groups of 25 male and 25 female Sprague-Dawley rats were given CGA185072 tech. in the diet at nominal concentrations of 0, 50, 500, 5000 and 10000 ppm continuously 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 100 day post weaning maturation period the selected F1 parental animals were mated and allowed to litter and to rear the F2a pups to weaning.

At 10000 ppm, effects in the P and Fl parental animals included reduced body weight and food consumption, the death of four Fl males and histological changes in the kidney and urinary bladder of the Fl animals. For pups, mean body weight was significantly reduced on day 21 for both F1a and F2a pups and the incidence of renal pelvic dilatation was increased in F1a pups. There were no adverse effects of 50, 500 or 5000 ppm CGA185072 tech.

There was no effect on fertility or general reproductive performance in either of the two generations at any dietary concentration of CGA185072 tech. in this study.