Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Mar - 12 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 1995
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FAT 40849/C TE
- Physical state: solid
- Colour: blue
- Analytical purity: 65.1% all coloured organic constituent, 43.6% main constituent
- Composition of test material, percentage of components: 65.1% all coloured organic constituent, 43.6% main constituent
- Purity test date: 20.08.2010
- Lot/batch No.: BOP 02-10
- Expiration date of the lot/batch: 15.08.2015
- Storage condition of test material: room temperature (20°C)
- Density: 1.738 g/cm³ at 19.7°C
- Solubility in Water: >473.7 g/L at 20°C

Test animals

Species:
rat
Strain:
other: Wistar Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: (P) 10-11 wks
- Weight at study initiation: (P) Males: 263.0-298.4 g; Females: 173.8-214 g
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (Lot. No. 081110)
- Diet: Altromin 1324 maintenance diet for rats and mice (Lot. No. 1307), ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark/hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The amount of test substance for each dose concentration was suspended separately in aqua ad injectionem (sterile water) on each administration day, immediately before the administration. Homogeneity was ensured by using a vortex machine.

VEHICLE
- Justification for use and choice of vehicle: Selection was based on the solubility of the test item
- Concentration in vehicle: 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no.: 0195A191
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- Females with unsuccessful mating were allowed to mate with other male of the same group.
- Females showing no evidence of copulation up to 14 day mating period were sacrificed 26 days after the last day of the mating period.
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each concentration was analysed for nominal concentration. Homogeneity in the vehicle was analysed for the low and high dose concentrations. Samples for nominal concentration verification were taken in Week 1 (first week of pre-mating period), Week 3 (first week of mating), Week 5 (gestation) and Week 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the low and high dose preparation in Week 1 and 5.
All concentration samples were stored frozen (approx. -20 °C) until the analysis was performed.
Duration of treatment / exposure:
Males: 14 days before mating, 14 days during mating (total 28 days of treatment)
Females: 14 days before mating, 14 days during mating, approx. 21-23 days during gestation, 3 days during lactation
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12-13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
other: yes, concurrent vehicle; the control group was shared with BSL Study no. 110996, another OECD 421 study, which was performed in parallel.
Details on study design:
- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. A descending sequence of dose levels was selected in order to demonstrate any dose-related response and a NOAEL.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily during weekend/holidays

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before assignment to the experimental groups and on the first day of administration.
Males were weighed weekly during the entire study period.
Females were weighed weekly during the pre-mating period, on Gestation day 0, 7, 14, 20 and on Post-natal day 0 (within 24 hours of parturition) and Post-natal day 4 along with pups.

FOOD CONSUMPTION:
- Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration and was not measured during the mating period.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring (on PND 0):
number and sex of pups, stillbirths, live births, runts, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS
yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after the completion of mating period (Day 29)
- Maternal animals: All surviving animals were sacrificed on respective PND 4.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively:
ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole)
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age (PND 4).

The animals were subjected to postmortem examinations for gross external abnormalities.
Statistics:
One-way analysis of variance (ANOVA) followed by DUNETT’s multiple comparison test (p<0.05 was considered as statistical significant)
Reproductive indices:
Copulation Index (%) = (No. of rats copulated /No. of pairs) x 100
Fertility Index (%) = (No. of females pregant/No. of females copulated) x 100
Delivery Index (%) = (No. of dams with live newborns/ No. of pregnant dams) x 100
Offspring viability indices:
Viability Index (%) = (No. of live offspring at Day 4/ No. of live offspring at birth) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no effects in males; 1000 mg/kg bw/day: 4 females: abnormal breathing, 2 females: salivation, 1 female: weight loss, 1 female: piloerection
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
no effects in both sexes throughout the study period; in high dose males statistical significant increase between pre-mating Days 7-14; in all females statistical significant decrease during gestation Day 7-14; the effects are considered non-adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
no effects in both sexes throughout the study period; in high dose males statistical significant increase between pre-mating Days 7-14; in all females statistical significant decrease during gestation Day 7-14; the effects are considered non-adverse.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
discoloration of various organs in all dose groups due to deposition of coloured test item (see details below)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
histological findings correlate to discoloration in kidney, lung and testis (see details below)

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
fertility index decreased in high dose group; copulation index unaffected; no effects on mean No. of corporea lutea, No. of implantation sites, No. of live pups born, %pre and post implantation loss, precoital interval and duration of gestation

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): In males, no treatment related clinical signs were observed. In females, abnormal breathing (4/10), salivation (2/10), weight loss (1/10) and piloerection (1/10) were observed in the 1000 mg/kg bw group. As these signs were observed only for few days in few animals they can not be conssidered as an adverse effect.
None of the animals died during the study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No test item-related effect on body weight was observed in both sexes throughout the complete study period. A statistically significant increase in body weight gain was observed in males of the 1000 mg/kg bw group in the pre-mating period (Days 7-14) and statistically significant decrease in females (all treatment groups) during gestation Day 7-14. No changes in food consumption were noted in the treated animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No treatment related effect on precoital interval and duration of gestation was observed.
The copulation index remained unaffected.
The fertility index was decreased to pregnancy rates of 60% in 1000 mg/kg bw group compared to 90% in the control group, 80% in 100 mg/kg bw group and 90% in the 300 mg/kg bw group.
No effects on mean No. of corporea lutea, No. of implantation sites, No. of live pups born, % pre and post implantation loss were observed.

ORGAN WEIGHTS (PARENTAL ANIMALS): No statistically significant changes in absolute and relative organ weights were observed.

GROSS PATHOLOGY (PARENTAL ANIMALS):
In males, red spots on right kidney (1/10 in control), discoloured/dark kidneys (9/10 in 100 mg/kg bw and 10/10 in 300 mg/kg bw), discoloured blue kidneys (10/10 in 1000 mg/kg bw), discoloured blue testes (10/10 in 1000 mg/kg bw), yellow spots on left epididymis (1/10 in control and 1/10 in 100 mg/kg bw), yellow spots on right epididymis (1/10 in 300 mg/kg bw) and discoloured blue epididymis (10/10 in 1000 mg/kg bw) were observed.
In females, discoloured dark kidneys (8/10 in 100 mg/kg bw, 10/10 each in 300 mg/kg bw and1000 mg/kg bw), hardening on the fat besides right ovary (1/10 in 100 mg/kg bw), discoloured dark ovaries (5/10 in 1000 mg/kg bw), discoloured dark uterus, oviduct and cervix (7/10 in 1000 mg/kg bw), discoloured dark vagina (3/10 in 1000 mg/kg bw), blue spots on the left side of the lung (1/10 in 1000 mg/kg bw) and blue spots on both sides (1/10 in 1000 mg/kg bw) were noted.
Blue discolouration/dark spots of the digestive tract (oesophagus to rectum) were seen in both sexes of all dose groups. The discolouration of organs is attributed to the colour of the test item and as such not a systemic effect due to the test item administration.

HISTOPATHOLOGY (PARENTAL ANIMALS): In the testis, greyish pigment in interstitial macrophages was observed in the majority of males treated at 1000 mg/kg/day. Basophilic granules were noted in a dose-related manner at 300 and 1000 mg/kg/day in the kidney, in the tubular epithelium of the cortex, in both sexes; in the females associated with golden-brown pigment deposition. In 1000 mg/kg bw females (3 animals), minor numbers of basophilic alveolar macrophages were seen in the lung. All changes were considered to be related to test item deposition. There was no histomorphological indication of functional impairment of the organs/tissues.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects NOAEL corresponding to the highest dose tested
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: copulation index; fertility index; delivery index

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Viability index unaffected. 7 pups from 1 mid dose female were found dead on PND 2.
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING): No treatment related effect was observed on total No. of pups born, No. of males, No. of females, sex ratio, live pups, still birth and runt on PND 0 and total No. of live pups and sex ratio on PND 4.
Viability index was unaffected. Survival of the pups from PND 0 - 4 remained unaffected in all treatment groups. However, 7 pups from 1 female of the 300 mg/kg bw group were found dead on PND 2.

BODY WEIGHT (OFFSPRING): No effect on group mean litter weight, total litter weight, male and female litter weight on PND 0 and PND 4.

GROSS PATHOLOGY (OFFSPRING): No substance related gross external findings were observed in any of the treated groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: gross pathology; litter weight; viability index; sex ratio

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Clinical Observations (P)

Clinical Findings

Control

100 mg/kg

300 mg/kg

1000 mg/kg

Males

Aggressive behavior

0/10

1/10

0/10

0/10

Alopecia

0/10

0/10

1/10

0/10

Females

Alopecia

1/10

0/10

1/10

0/10

Moving the bedding

0/10

0/10

2/10

0/10

piloerection

0/10

0/10

1/10

1/10

dehydration

0/10

0/10

1/10

0/10

abnormal breathing

0/10

0/10

0/10

4/10

salivation

0/10

0/10

0/10

2/10

weight loss

0/10

0/10

0/10

1/10

Table 2: Macroscopic Findings (P)

Findings (External/Internal)

Control

100 mg/kg

300 mg/kg

1000 mg/kg

Males

kidney (right): red spots

1/10

0/10

0/10

0/10

kidneys: discoloured dark

0/10

9/10

10/10

0/10

kidneys: discoloured blue

0/10

0/10

0/10

10/10

epididymis (left): yellow spot

1/10

1/10

0/10

0/10

epididymis (right): yellow spot

0/10

0/10

1/10

0/10

epididymis: discoloured blue

0/10

0/10

0/10

10/10

testes: discoloured blue

0/10

0/10

0/10

10/10

pancreas, stomach, duodenum, jejunum, ileum, caecum, colon: discoloured blue

0/10

0/10

0/10

1/10

Females

kidneys: discoloured dark

0/10

8/10

10/10

10/10

hardening on the fat besides right ovary

0/10

1/10

0/10

0/10

ovaries: discoloured dark

0/10

0/10

0/10

5/10

uterus, oviduct, cervix: discoloured dark

0/10

0/10

0/10

7/10

lung: left side blue spot

0/10

0/10

0/10

1/10

lung: both sides blue spots

0/10

0/10

0/10

1/10

vagina: discoloured dark

0/10

0/10

0/10

3/10

 

Table 3: Body weight changes

Day of treatment

Group

 

Control

100 mg/kg bw

300 mg/kg bw

1000 mg/kg bw

Males

Pre-mating

1-7

Mean

19.10

19.80

22.10

22.90

SD

2.73

4.47

7.03

7.85

7-14

Mean

13.50

13.50

15.70

20.30*

SD

4.60

3.63

3.53

7.48

Mating/Post-mating

14-21

Mean

11.40

13.60

14.30

13.50

SD

2.59

4.12

4.06

5.74

21-28

Mean

10.60

11.70

10.80

10.80

SD

3.50

4.27

4.69

5.09

28-TS

Mean

3.50

2.70

-0.20

4.20

SD

2.37

2.36

5.65

2.97

1-TS

Mean

58.10

59.49

62.70

71.70

SD

11.01

8.01

13.37

24.68

N

10

10

10

10

Females

Pre-mating

1-7

Mean

3.50

5.20

4.10

6.60

SD

6.31

5.39

5.78

4.43

7-14

Mean

7.40

9.00

7.20

7.10

SD

4.14

3.46

3.97

3.18

 

N

10

10

10

10

Gestation

0-7

Mean

24.22

21.75

21.44

18.50

SD

6.02

4.37

5.96

3.94

7-14

Mean

31.22

26.00

30.33

22.17*

SD

4.63

4.78

3.81

11.79

14-20

Mean

60.78

57.00

46.78

50.00

SD

11.78

9.09

20.97

14.52

0-20

Mean

116.22

104.75

98.56

90.67

SD

17.47

14.93

26.06

26.69

N

9

8

9

6

Lactation

0-4

Mean

4.00

9.50

3.44

5.33

 

SD

5.85

6.61

9.93

6.50

 

N

9

8

9

6

TS - Terminal sacrifice

*Significant (p<0.05); as determined with the individual data

Table 4: Reproductive Indices

Index

Group

 

Control

100 mg/kg bw

300 mg/kg bw

1000 mg/kg bw

Copulation Index

%

100

100

100

100

Fertility Index

%

90

80

90

60

Delivery Index

%

100

100

100

100

Viability Index

Mean

100.00

100.00

92.93

100.00

SD

0.00

0.00

21.21

0.00

N

9

8

9

6

Applicant's summary and conclusion

Conclusions:
The test substance had no effect on reproductive performance.
CLP: not classified
DSD: not classified
Executive summary:

A reproduction/developmental toxicity screening test of FAT 40849/C TE was conducted in Wistar rats to assess possible effects on the fertility and embryofetal development.

Four groups comprising of 10 male and 10 non-pregnant nulliparous female rats each were dosed daily by gavage with 100, 300 and 1000 mg/kg bw/day of FAT 40849/C TE. The control group received sterile water in similar volume as the treated groups. Administration was carried out during 14 days pre-mating and 14 days mating period in both males and in females, during gestation period and up to Post natal day (PND) 3 in females. Males were dosed for 28 days. Males and females were sacrificed on Day 29 and PND 4, respectively and subjected to necropsy.

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed on PND 1 and 4.

No mortalities or predominant clinical signs considered to be treatment-related were observed during the study period. Body weight and food consumption were as well not affected by the treatment, as were precoital interval, duration of gestation and reproductive indices. Discolouration of organs and the digestive tract noted in almost all treated animals is considered to be attributable to the colour of the test item and as such not a systemic effect due to the test item administration. No treatment-related or dose-dependent effect regarding organ weight was seen in either sex or dose group. Histological findings were considered to be related to test item deposition with no accompanying findings that would indicate organ damage.

No treatment-related effect was observed regarding any litter parameter examined.

Based on the data generated from this reproduction/developmental toxicity screening test with FAT 40849/C TE, the NOAEL is considered to be 1000 mg/kg bw/day in males and females.