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

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Data is from peer-reviewed journal.

Data source

Reference

Reference Type:

publication

Title:

Studies on the Teratogenicity of Food Additives (4) Effects of Food Dye on the Preand Postnatal Development in Rats in Relation to Fetal Distribution

Author:

Shinsuke NAKAURA,

Year:

1975

Bibliographic source:

J. Food Hyg. Soc. 1975

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The study was conducted to determine the reproductive toxicity potential of 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen -9-yl)benzoic acid (Food dye Red No.104 / phloxine) (CAS No.- 18472-87-2) when administered orally to wistar pregnant rats.

GLP compliance:

not specified

Limit test:

no

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:

Sodium 2,3,4,5-tetrachloro-6-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate

Test material form:

solid: particulate/powder

Details on test material:

IUPAC name: 3,4,5,6-Tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid
Molecular weight: 829.6388
Molecular formula: C20-H2-Br4-Cl4-O5.2Na
Smiles: [Na+].[Na+].[O-]c1c(Br)cc2c(Oc3c(Br)c([O-])c(Br)cc3C24OC(=O)c5c(Cl)c(Cl)c(Cl)c(Cl)c45)c1Br
Inchl: InChI=1S/C20H4Br4Cl4O5.2Na/c21-5-1-3-17(9(23)15(5)29)32-18-4(2-6(22)16(30)10(18)24)20(3)8-7(19(31)33-20)11(25)13(27)14(28)12(8)26;;/h1-2,29-30H;;/q;2*+1/p-2

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

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Nihon Rat Co. Ltd., Tokyo.
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation:207.1-211.3g
- Fasting period before study: No data available
- Housing: Housed individually
- Diet (e.g. ad libitum): basal laboratory chow (NMF,Oriental Yeast Co., Tokyo) ad libitum
- Water (e.g. ad libitum): No data available
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 2 degC
- Humidity (%): 50-60 %
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: basal laboratory chow

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Phloxine certified for food dye was obtained as powder from Hodogaya Chem. Co. Phloxine diet was prepared to contain 0, 0.3, 1.0 and 3.0% in a basal laboratory chow.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal laboratory chow
- Concentration in vehicle: 0, 0.3, 1.0 and 3.0% (0, 280, 920, 2870 mg/kg/day)
- 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: No data available
- Length of cohabitation: Overnight
- Proof of pregnancy: Next morning those with vaginal plug or sperm in the vaginal smear were taken to be in day zero 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 data available
- After successful mating each pregnant female was caged (how): No data available
- Any other deviations from standard protocol: No data available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

19 days

Frequency of treatment:

Daily

Details on study schedule:

Food dye Red No.104(phloxin) was administered to Wistar pregnant rats at levels of 0, 0.3, 1, and 3% in diet during pregnancy and teratogenic effects and fetal and maternal organ distribution was examined. Suppression in maternal body weight gain and growth retardation in foetuses was observed in the highest dose group. No evidences of increase in fetal death and malformation were obtained in all groups. The postnatal development was maintained well without any adverse effects. However all newborn from 4 out of 5 dams in 3% group was killed by cannibalism within 2 days after birth.

Doses / concentrations

Remarks:

0, 0.3, 1.0 and 3.0% (0, 280, 920, 2870 mg/kg/day)

No. of animals per sex per dose:

Total animals – females 80
0%(0 mg/kg/day): 20 female
0.3%(280 mg/kg/day): 20 female
1%(920 mg/kg/day): 20 female
3%(2870 mg/kg/day): 20 female

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Clinical sign, body weight and body weight gain, Food and Water Consumptions and food efficiency was examined.

Oestrous cyclicity (parental animals):

Any irregularity in estrous cyclicity was observed. Number of corpora lutea, inplantations and rate of nidations are also examined.

Sperm parameters (parental animals):

No data available

Litter observations:

Fetal mortality, litter size, body weight, body length, fetal resorption, sex ratio and tail length was observed. Motor activities, righting reflex, pinna reflex, pain response and startle response were also examined.

Postmortem examinations (parental animals):

Organ weight and gross pathology were observed.

Postmortem examinations (offspring):

Organ weight, visceral and skeletal anomalies, internal organ anomalies was examined.

Statistics:

No data available

Reproductive indices:

Gestation index, total implants index, birth index, weaning index and delivery index were examined

Offspring viability indices:

Viability in day 0, 4 and 8 week were examined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No toxic symptoms were observed in treated dams as compared to control.

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the highest dose level of 3%, a slight depression of weight gain were observed, but any other toxic symptoms were not observed throughout the period of pregnancy. No marked changes in 0.3 and 1% groups were noted.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No effect was observed in food consumption of treated rats as compared to control.
The net amounts of phloxine ingested daily during pregnancy were 2.87g/kg for 3% group, 0.92 g/kg for 1% group and 0.28g/kg for 0.3% group, respectively.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

At the highest dose level of 3%, decrease in food efficiency were observed.

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:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No significant effect were observed in the number of corpora lutea or implantations and the rate of nidation of treated female rats as compared to control.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

920 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

food efficiency

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

other: No adverse effects was observed.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

not measured/tested

Target system / organ toxicity (P1)

Critical effects observed:

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 evidences of increase in fetal mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly decrease in body weight was observed in female offspring at 2870 mg/kg/day dose groupe as compared to control.

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:

effects observed, treatment-related

Description (incidence and severity):

Significant effect were observed in lung weight of male rats at 920 mg/kgbw/day as compared to control. The observed changes were not remarkable.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological anomalies and skeleton were observed in treated offsprings as compared to control.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

No evidences of fetuses with external and internal malformations were obtained in all groups.
Prenatal development In 2870 mg/kg bw/day, slight increase in nucleus with deformed shapes and low stainability insternebrae and decreases in the number of metacarpus and coccygeal vertebrae were observed. These findings may suggest a retardation of ossification in fetal stage, but such changes were not observed in rats examined after weaning.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

No significant difference between the phloxine-treated and control groups was found in the mean litter size, delivering rate or body weight of newborn at birth. In 3% group, however, all newborn from 4 out of 5 dams examined were killed by cannibalism of the dams within 2 days after birth. The mean litter sizes at the 4th and 8th weeks after birth were almost same among 0.3%, 1% and control groups. With the female of 3% group, significantly low body weight was obtained, but no appreciable failures.

Body lenght: Decrase in body lenght was observed in 2870 mg/kg/day treated offsprings as compared to control.

Tail lenght: No effect were observed on tail lenght of treated offspring as comparted to control.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Neurobehavioural parameters: No effect were observed on motor activities, righting reflex, pinna reflex, pain response and startle response of treated offsprings as compared to control.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

The postnatal development was maintained well without any adverse effects.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

920 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect on survival, body weight, body lenght, tail lenght, organ weight, gross pathology and histopathology

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

Effect levels (F2)

Remarks on result:

not measured/tested

Target system / organ toxicity (F2)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 920 mg/kg/day for F0 and F1 generation when Wistar female rats treated with 3,4,5,6-tetrachloro-2- (1,4,5,8-tetra bromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Food dye Red No.104 / phloxine) (CAS No.18472-87-2).

Executive summary:

In a one generation reproductive toxicity study, wistar female rats treated with 3,4,5,6-tetrachloro-2- (1,4,5,8-tetra bromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (Food dye Red No.104 / phloxine) (CAS No.18472-87-2) in the concentrations of 0, 280, 920, 2870 mg/kg/day (0, 0.3, 1.0 and 3.0 %) during gestation orally by feed. No toxic symptoms were observed in treated rats. No effect on food consumption and water consumptions were observed in treated rats. Slight decrease in body weight gain and decrease in food efficiency and decrase in body lenght was observed in 2870 mg/kg/day treated offsprings as compared to control.No significant effects were observed in number of corpora lutea or implantations and the rate of nidation, organ weights and gross pathology in treated female rats as compared to control. No effect were observed on litter size and fetal mortality. Significantly decrease in body weight was observed in female offspring at 2870 mg/kg/day. Significant effect were observed in lung weight of male rats at 920 mg/kgbw/day as compared to control.The observed change in lung weight were not remarkalbe. No effect on tail lenght and gross pathology were observed in offspring. During prenatal development , slight increase in nucleus with deformed shapes and low stainability in sternebrae and decreases in the number of metacarpus and coccygeal vertebrae were observed. These findings may suggest a retardation of ossification in fetal stage, but such changes were not observed in rats examined after weaning.Therefore, NOAEL was considered to be 920 mg/kg/day for F0 and F1 generation when Wistar female rat treated with 3,4,5,6-tetrachloro-2- (1,4,5,8-tetra bromo-6-hydroxy-3- oxoxanthen-9-yl)benzoic acid (Food dye Red No.104 / phloxine) (CAS No.18472-87-2) orally by feed.