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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed journal

Data source

Reference
Reference Type:
publication
Title:
A TERATOGENICITY STUDY OF PHLOXINE B IN ICR MICE
Author:
M. SENO, S. FUKUDA and H. UMISA
Year:
1984
Bibliographic source:
Fd Chem. Toxic. Vol. 22, No. 1 pp. 55-60, 1984

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
one-generation toxicity study of Phloxine B in Jcl:ICR Mice
GLP compliance:
not specified
Limit test:
no
Justification for study design:
No data available

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): PHLOXINE B
- Molecular formula (if other than submission substance): C20H4Br4Cl4O5.2Na
- Molecular weight (if other than submission substance): 829.64 g/mol
- Substance type: Organic
- Physical state: Red-brown powder
- Impurities (identity and concentrations): < 5 ( weight loss on drying < 10%; chloride and sulphate < 5% )
Specific details on test material used for the study:
- Name of test material (as cited in study report):PHLOXINE B
- Molecular formula :C20H4Br4Cl4O5.2Na
- Molecular weight :829.64 g/mol
- Substance type: Organic
- Physical state: Red-brown powder
- Impurities (identity and concentrations): < 5 ( weight loss on drying < 10%; chloride and sulphate < 5% )

Test animals

Species:
mouse
Strain:
other: Jcl:ICR
Details on species / strain selection:
No data available
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Nippon CLEA Co., Ltd, Tokyo.
- Age at study initiation: 8-9 wk old)
- Weight at study initiation:
(P) 26--31 g- female, male - No data available.
(F1)- No data available
. - Fasting period before study: No data available.
- Housing: Males were housed singly and females four to a cage.
- Diet (e.g. ad libitum): commercial diet
(Nippon CLEA, CE-2) ad lib.
- Water (e.g. ad libitum): tap-water ad lib.
- Acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 + I°C
- Humidity (%):55 + 5%
- Air changes (per hr): No data available.
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle

IN-LIFE DATES: From: To No data available.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Nippon CLEA, CE-2
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: No data available

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): Nippon CLEA, CE-2 commercial diet
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data available
- Concentration in vehicle: 0, 1.0, 3.0 and 5.0%
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available
Details on mating procedure:
- M/F ratio per cage:1:1
- Length of cohabitation:Overnight.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Females with vaginal plugs were considered to be in day 0 of pregnancy.
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility.No data available
- Further matings after two unsuccessful attempts: [no / yes (explain)]No data available
- After successful mating each pregnant female was caged (how):Females were housed individually.
- Any other deviations from standard protocol:No data available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
11 day (6-16 day of gestation)
Frequency of treatment:
daily
Details on study schedule:
Pregnant Jcl:ICR mice were given Phloxine B in the diet at concentrations of 0, 1, 3 and 5% from the morning of day 6 through day 16 of gestation. The mice were killed on day 18 and fetuses were examined for external, visceral and skeletal anomalies.
Doses / concentrations
Remarks:

0, 1.0, 3.0 and 5.0%(2000,6000 and 10000 mg/kg/day)

No. of animals per sex per dose:
Total No – 84 Female
0 % (0mg/kg/day)22 females
1%(2000 mg/kg/day)21 females
3%(6000 mg/kg/day)20 females
5% (10000 mg/kg/day)21 females
Control animals:
yes, concurrent vehicle
Details on study design:
No data available
Positive control:
No data available

Examinations

Parental animals: Observations and examinations:
Parental animal: observation and examination:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Dams were weighed on days 6, 12, 16 and 18.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OTHER:
Estrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
No data available
Litter observations:
Foetal body weight was observed
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]yes on day 18 the mice were killed under ether anaesthesia

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]Absolute and Relative organ weight, numbers of corpora lutea, implantations or live fetuses were observed.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]Fetuses were observed for soft tissue abnormalities and skeletal anomalies.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
The litter was considered to be the experimental unit for the analysis of data on embryo/foetal toxicity and teratogenicity. Statistical significance of the differences between groups was determined by the Mann-Whitney U test (Siegel, 1956). Two-tailed tests were performed and P < 0.05was selected as the level of statistical significance
Reproductive indices:
Number of females with live fetuses, resorptions, Number of dead fetuses and total resorptions were examined.
Offspring viability indices:
No data available

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No overt signs of toxicity were observed in treated dams.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In 10,000 mg/kgbw/day, 2 dams died on days 16 and 17. Another female in this group
aborted on day 17.These three animals were excluded from all computations.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased in Maternal body-weight gains for days 6-16 of gestation were observed in 2000, 6000 and 10,000 mg/kg bw/day treated dams as compared to control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Average food intakes of the groups were approximately equal both in treated and control group.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The maternal liver weight, expressed as either the absolute or the relative weight, was significantly increased among dams in the 10000mg/kg bw dose group, but not among those receiving the 2000mg/kg bw dose group when compared with the control.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

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):
The numbers of corpora lutea, implantations or live foetuses in all of the treated groups were slightly decreased, but not significantly in comparison with those of the control. No effect were observed on Number of dead fetuses, Total resorptions and Number of live fetuses of treated rats as compared to control.

Details on results (P0)

Mortality:
In 10,000 mg/kgbw/day, 2 dams died on days 16 and 17. Another female in this group
aborted on day 17.

These three animals were excluded from all computations.

Clinical signs: No overt signs of toxicity were observed in treated dams.

Body weight:
Significantly decreased in Maternal body-weight gains for days 6-16 of gestation were observed in 2000, 6000 and 10,000 mg/kg bw/day treated dams as compared to control.

Food consumption: Average food intakes of the groups were approximately equal both in treated and control group.

Test substance intake: No data available

Reproductive function: estrous cycle: No data available

Reproductive function: sperm measures: No data available

Reproductive performance: The numbers of corpora lutea, implantations or live foetuses in all of the
treated groups were slightly decreased, but not significantly in comparison with those of the control.
No effect were observed on Number of dead fetuses, Total resorptions and Number of live fetuses of treated rats as compared to control.

Gross pathology: No data available

Histopathology: No data available

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effect on survival, clinical signs, body weight, food consumption, organ weight and reproductive performance
Remarks on result:
other: No effects on reproductive performance
Dose descriptor:
LOAEL
Effect level:
6 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: toxic effects was observed

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No effect were observed on number of live fetuses in treared rats
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Foetuses with open eyelids were observed in all treated dams. Exencephaly, significantly increased incidence of cleft palate were observed in 10,000 mg/kgbw/day treated fetuses as compaed to control. These types of external abnormalities have been seen spontaneously in this strain.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased numbers of ossified caudal vertebrae and phalanges, slightly reduced incidence of accessory sternebrae and significantly increased numbers of foetuses with a fourteenth rib or with an extra rib (at least one fourteenth rib that was half or greater than half the length of the preceding rib) were observed in 10,000 mg/kgbw/day dose group.
Other effects:
not specified
Description (incidence and severity):
Increase in fourteenth or extra ribs might be taken as an indicator of the possible teratogenic potential of a chemical.
Slight retardation of ossification were observed in 6000 mg/kgbw/day treaed fetuses as compared to control.
Splitting of the cervical vertebral arches were observed in 2000 mg/kg bw/day treatet fetouses.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

Mortality:
No effect were observed on number of live fetuses in treared rats

Clinical signs: No data available

Gross pathology: Foetuses with open eyelids were observed in all treated dams.
Exencephaly, significantly increased incidence of cleft palate were observed in 10,000 mg/kgbw/day treated fetuses as compaed to control.
These types of external abnormalities have been seen spontaneously in this strain.

Histopathology: Significantly decreased numbers of ossified caudal vertebrae and phalanges, slightly reduced incidence of accessory sternebrae and significantly increased numbers of foetuses with a fourteenth rib or with an extra rib (at least one fourteenth rib that was half
or greater than half the length of the preceding rib)
were observed in 10,000 mg/kgbw/day dose group.

Increase in fourteenth or extra ribs might be taken as an indicator of the possible teratogenic potential of a chemical.

Slight retardation of ossification were observed in 6000 mg/kgbw/day treaed fetuses as compared to control.

Splitting of the cervical vertebral arches were observed in 1000 mg/kg bw/day treatet fetouses.

Effect levels (F1)

Dose descriptor:
LOAEL
Generation:
F1
Effect level:
2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Adverse effect on gross pathology and histopathology
Remarks on result:
other: Developmental effects was observed

Target system / organ toxicity (F1)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Maternal and reproductive data for mice given 0-5% Phloxine B in the diet during days 6-16 of gestation

Parameters

group

Value in control or treated group

Control

1%

3%

5%

No. of pregnant females examined

 

22

21

20

21

No. of females with live foetuses

 

22

21

20

18+

Maternal body-wt gain (g, days 6-16)

 

20.5

18.5*

18.2*

17.1*

No. of corpora lutea/dam

 

14.8

13.7

14.1

14.3

No. of implantations/dam

 

13.9

12.8

12.4

13.0

No. of resorptions/dam

 

1.0

1.4

0.7

1.1

No. of dead foetuses/dam

 

0.1

0.1

0.3

0.2

Total resorptions (%)

 

8.0

11.7

8.2

9.5

No. of live foetuses/litter

 

12.8

11.3

11.4

11.8

Total no. of live foetuses (male/female)

 

136/145

114/124

117/111

108/104

Mean foetal body weight (g)

 

1.42

1.44

1.43

1.42

Maternal liver weight

 

 

 

 

 

Absolute (g)

 

3.04

2.88

3.12

3.47*

Relative (g/100 g body wt)

 

10.7

10.2

11.0

12.2

+One dam aborted and two dams died before day 18 of gestation. These three dams were excluded from all computations.

Values marked with asterisks are significantly different from the corresponding control value (*P < 0.05;Mann-Whitney U test).

Incidence of external and soft-tissue anomalies in foetuses of mice given 0-5% Phloxine B in the diet during days 6-16 of gestation

 

Anomalies

group

No. of foetuses (litters) affected

Control

1%

3%

5%

External anomalies

No. of foetuses (litters) examined

 

281(22)

238(21)

228(20)

212(18)

No. abnormal

8(7)

4(3)

7(4)

11(6)

Open eyelids

7(6)

4(3)

7(4)

1(1)

Exencephaly

1(1)+

0

0

1(1)

Cleft palate

1(1)

0

0

9(5)*

Soft-tissue anomalies

No. of foetuses (litters) examined**

 

102(22)

86(21)

89(20)

77(18)

+ This anomaly occurred in a foetus affected with open eyelids.

**None of the foetuses examined showed any visceral anomalies.

Values marked with asterisks are significantly different from the corresponding control

Mann-Whitney U test).

Skeletal data for foetuses of mice given 0-5% Phloxine B in the diet during days 6-16 of gestation

 

Observation

group

Incidence +

 

control

1%

2%

3%

No. of foetuses examined

 

179(22)

152(21)

139(20)

135(18)

No. of ossified caudal vertebrae per foetus

9.71

9.39

9.63

8.25*

No. of ossified phalanges in forelimbs per foetus

10.2

9.74

10.0

7.85*

No. of ossified phalanges in hindlimbs per foetus

10.7

10.1

10.3

7.53*

Accessory sternebrae

26(11)

16(12)

21(12)

9(8)

Cervical rib

18(11)

14(8)

25(10)

24(12)

Fourteenth rib

67(21)

76(18)

68(17)

112(18)*

Extra rib**

33(14)

39(15)

47(15)

93(18)*

Skeletal anomalies

No. abnormal

 

1(1)

3(3)

14(9)*

42(14)*

Fused ribs

1(1)

1(1)

0

0

Fusion of the cervical vertebral arches

0

0

0

2(2)+0

 

 

 

 

 

Fusion of the lumber vertebral bodies

0

0

0

1(1)

Splitting of the thoracic vertebral arche

0

0

0

3(2)

Splitting of the cervical vertebral arches (nos 3-6)

 

0

2(2)

14(9)*

37(14)**

 

 

+Where appropriate, this is given as the no. of foetuses affected and, in parentheses, the no. of litters containing affected foetuses.

*Extra rib was defined as at least one 14th rib that was half or greater than half the length of the preceding rib.

**One anomaly occurred in a foetus affected with splitting of the cervical vertebral arches

Numbers marked with asterisks are significantly different from the corresponding control number (*P < 0.05,Mann-Whitney U test on litter data

Applicant's summary and conclusion

Conclusions:
NOAEL was considered to be 2000 mg/kg body weight/day (1%) for F0 generation and LOAEL was considered to be 2000 mg/kg body weight/day (1%) for F1 genration when Jcl:ICR female mice treated with Phloxine B.
Executive summary:

In a reproductive and developmental toxicity study, Jcl:ICR female mice treated with Phloxine B(18472 -87-2). 84 females mice were used in the study.The female were caged with male in pairs overnight. The next morning female with vaginal plugs were considered to be in day 0 of pregnancy and were housed individually. The test material administered in the concentration of 0, 2000,6000 and 10000 mg/kg/day (0, 1.0, 3.0 and 5.0 %)from the morning of day 6 through day 16 of pregnancy by oral feed. Animals were observed daily, beginning on day 6 of pregnancy, and were weighed on days 6, 12, 16 and 18. Food consumption and spillage were measured at regular intervals beginning on day 6 of pregnancy, and net amounts of food ingested were calculated. On day 18, the mice were killed under ether anaesthesia, and the maternal liver weight was recorded. Their reproductive status was determined. Corpora lutea were counted under a dissecting microscope. Implantation sites in each uterine horn were counted and the general condition of each conceptus was recorded. The live foetuses were removed, weighed, sexed and examined for external malformations. A third of the foetuses in each litter (selected randomly) were fixed in Bouin's solution and examined for soft-tissue abnormalities by a modified razor-section technique.The remainder were fixed in 95%ethanol, cleared with 1% KOH, stained with Alizarin Red S (Dawson, 1926) and examined for skeletal alteration.2 dams died on days 16 and 17. Another female in this group aborted on day 17.Significantly decreased in Maternal body-weight gains for days 6-16 of gestation were observed in 2000, 6000 and 10,000 mg/kg bw/day treated dams as compared to control. Similarly, the numbers of corpora lutea, implantations or live foetuses in all of the treated groups were slightly decreased, but not significantly in comparison with those of the control. Absolute and relative liver weight was significantly increased in 2000 mg/kg bw/day dose group as compared to control in F0 generation. In addition, Foetuses with open eyelids were observed in all treated dams. Exencephaly, significantly increased incidence of cleft palate and Significantly decreased numbers of ossified caudal vertebrae and phalanges, slightly reduced incidence of accessory sternebrae and significantly increased numbers of foetuses with a fourteenth rib or with an extra rib (at least one fourteenth rib that was half or greater than half the length of the preceding rib) were observed in 10,000 mg/kgbw/day treated fetuses as compared to control. Slight retardation of ossification was observed in 6000 mg/kgbw/day treated foetuses as compared to control. Splitting of the cervical vertebral arches was observed in 1000 mg/kg bw/day treated foetuses as compared to control. Hence, dose response was seen in the total incidence of skeletal abnormalities, suggested a teratogenic effect. Therefore, NOAEL was considered to be 2000 mg/kg body weight/day (1%) for F0 generation and LOAEL was considered to be 2000 mg/kg body weight/day (1%) for F1 generation when Jcl:ICR female mice were treated with Phloxine B orally by feed from day 6 to 16 of gestation.