3,4,5,6-tetrachloro-2-(2,4,5,7-tetrabromo-3,6-dihydroxyxanthen-9-yl)benzoic acid, aluminium salt

Toxicity to reproduction

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

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

Test material information

Constituent 1

Reference substance name:

3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid

EC Number:

242-355-6

EC Name:

3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid

Cas Number:

18472-87-2

Molecular formula:

C20H4Br4Cl4O5.2Na

IUPAC Name:

2',4',5',7'-tetrabromo-4,5,6,7-tetrachloro-3',6'-dihydroxy-3H-spiro[2-benzofuran-1,9'-xanthen]-3-one

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 or test system 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:

Oestrous 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 generation)

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

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous 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)

1

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

2

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.