Sodium 3-[(1,5-dihydroxy-2-naphthyl)azo]-4-hydroxybenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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

Principles of method if other than guideline:

See attachment

GLP compliance:

not specified

Limit test:

no

Justification for study design:

The test substance was administered in diet to 60 mice (10/sex/group) at dietary levels of 0.42, 0.84, and 1.68 %. The control groups (20 mice, 10/sex) were given the basal diets (Nihon Clea, CE-2) for the corresponding period. Individual food intake of mice was measured during 5 divided periods; pre-conception (from 5 weeks of age to mating), mating (5 days), gestation (14 days), and lactation (from birth to weaning), and F 1 generation (4-9 weeks of age).
The animals from the F0 generation were 5 weeks of age at the start of the study.
At 9 weeks of age, each female was paired with one male from the same treatment group, for a period of 5 days. The males were removed from the females after 5 days, and the females were allowed to carry their litters to term, deliver and rear their offspring.
In the F 1 generation, litter size, litter weight and sex ratio (male/female) were measured on postnatal day (PND) 0 (at birth). The offspring were individually weighed on PNDs 0, 4, 7, 14 and 21 during the lactation period. The offspring were weaned when they were 4 weeks of age, and were randomly selected to continue treatment, one male and one female from each litter.

Neurobehavioral procedure
The functional and behavioral developmental parameters were measured and scored for individual offspring during the lactation period in the Fl generation (Tanaka, 1992), and were analysed on a whole-litter basis (Abbey and Howard, 1973). The measured parameters were as follows: surface righting on PNDs 4 and 7 (Fox, 1965; Pantaleoni et al., 1988), negative geotaxis on PNDs 4 and 7 (Fox, 1965; Altman and Sudarshan, 1975; Pantaleoni et al., 1988), cliff avoidance on PND 7 (Fox, 1965; Altman and Sudarshan, 1975; Pantaleoni et al., 1988), swimming behavior (direction, head angle, and limb movement) on PNDs 4 and 14 (Fox, 1965; Pantaleoni et al., 1988), and olfactory orientation on PND 14 (Altman and Sudarshan, 1975; Barlow et al., 1978; Meyer and Hansen, 1980).
Exploratory behavior of mice was measured in an animal movement analysing system ANIMATE AT-420 (Toyo Sangyo Co., Ltd, Toyama, Japan) at 3 and 8 weeks of age in the F1 generation. The system consisted of a doughnut-shaped cage with 36 detectors for measuring spontaneous motor activity (Matsumoto et al., 1990a, b).
The behavioral parameters were recorded for 5 min on one male and one female selected randomly from each litter at 3 weeks of age, and for 10 min on all animals at 8 weeks of age. The measurement parameters were number of movements, movement time (s), number of horizontal activities, total distance (cm), number of vertical activities, vertical time (s), number of turnings, average distance (cm), average speed (cm/s), and defecations.
The animals performed 3 trials in a multiple water T-maze of Biel's type adapted for mice at 7 weeks of age in the FI generation (Biel, 1940; Kitatani et al., 1988). The water temperature was maintained at 20 ± 1 °C. The time taken and number of errors were measured from the start to finish for a maximum 120 s. If the time taken was greater than 120 s, it was recorded as 120 s (Kitatani et al., 1988).

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female mice (Crj: CD-1, 4 weeks of age) were purchased from Charles River Japan Inc., Kanagawa, Japan.
They were individually housed in polycarbonate solid-floored cages with wood flakes, and kept in a temperature controlled room maintained at 24 +/- 1°C with a relative humidity of 55 +/- 5% and a 12-h light/dark cycle. They were given control or experimental diets (Nihon Clea, CE-2) and water ad libitum.

Route of administration:

oral: feed

Details on exposure:

Allura Red AC was administered in diet to 60 mice (10/sex/group) at dietary levels of 0.42, 0.84, and 1.68 %.
The control groups (20 mice, 10/sex) were given the basal diets (Nihon Clea, CE-2) for the corresponding period.
Individual food intake of mice was measured during 5 divided periods; pre-conception (from 5 weeks of age to mating), mating (5 days), gestation (14 days), and lactation (from birth to weaning), and F 1 generation (4-9 weeks of age).

Details on mating procedure:

The animals from the F0 generation were 5 weeks of age at the start of the study.
At 9 weeks of age, each female was paired with one male from the same treatment group, for a period of 5 days.
The males were removed from the females after 5 days, and the females were allowed to carry their litters to term, deliver and rear their off
spring.
In the F1 generation, litter size, litter weight and sex ratio (male/female) were measured on postnatal day (PND) 0 (at birth).

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

F0: from 5 weeks of age during mating (f+m), gestation and lactation (f)
pre-conception (from 5 weeks of age to mating), mating (5 days), gestation (14 days), and lactation (from birth to weaning), and
F1 generation: 4-9 weeks of age

Frequency of treatment:

continuous

Details on study schedule:

The animals from the F0 generation were 5 weeks of age at the start of the study.
At 9 weeks of age, each female was paired with one male from the same treatment group, for a period of 5 days. The males were removed from the females after 5 days, and the females were allowed to carry their litters to term, deliver and rear their offspring.

In the F1 generation, litter size, litter weight and sex ratio (male/female) were measured on postnatal day (PND) 0 (at birth). The offspring were individually weighed on PNDs 0, 4, 7, 14 and 21 during the lactation period. The offspring were weaned when they were 4 weeks of age, and were randomly selected to continue treatment, one male and one female from each litter.

The functional and behavioral developmental parameters were measured and scored for individual offspring during the lactation period in the F1 generation (Tanaka, 1992), and were analysed on a whole-litter basis (Abbey and Howard, 1973). The measured parameters were as follows: surface righting on PNDs 4 and 7 (Fox, 1965; Pantaleoni et al., 1988), negative geotaxis on PNDs 4 and 7 (Fox, 1965; Altman and Sudarshan, 1975; Pantaleoni et al., 1988), cliff avoidance on PND 7 (Fox, 1965; Altman and Sudarshan, 1975; Pantaleoni et al., 1988), swimming behavior (direction, head angle, and limb movement) on PNDs 4 and 14 (Fox, 1965; Pantaleoni et al., 1988), and olfactory orientation on PND 14 (Altman and Sudarshan, 1975; Barlow et al., 1978; Meyer and Hansen, 1980).
Exploratory behavior of mice was measured in an animal movement analysing system ANIMATE AT-420 (Toyo Sangyo Co., Ltd, Toyama, Japan) at 3 and 8 weeks of age in the F1 generation. The system consisted of a doughnut-shaped cage with 36 detectors for measuring spontaneous motor activity (Matsumoto et al., 1990a, b).
The behavioral parameters were recorded for 5 min on one male and one female selected randomly from each litter at 3 weeks of age, and for 10 min on all animals at 8 weeks of age. The measurement parameters were number of movements, movement time (s), number of horizontal activities, total distance (cm), number of vertical activities, vertical time (s), number of turnings, average distance (cm), average speed (cm/s), and defecations.
The animals performed 3 trials in a multiple water T-maze of Biel's type adapted for mice at 7 weeks of age in the FI generation (Biel, 1940; Kitatani et al., 1988). The water temperature was maintained at 20 ± 1 °C. The time taken and number of errors were measured from the start to finish for a maximum 120 s. If the time taken was greater than 120 s, it was recorded as 120 s (Kitatani et al., 1988).

Dose / conc.:

0.42 other: %

Remarks:

in diet

Dose / conc.:

0.84 other: %

Remarks:

in diet

Dose / conc.:

1.68 other: %

Remarks:

in diet

Remarks:

Doses / Concentrations:
700, 1420, 2928 mg/kg/day
Basis:
nominal in diet
males, pre-conception

Remarks:

Doses / Concentrations:
803, 1694, 3375 mg/kg/day
Basis:
nominal in diet
females, pre-conception

Remarks:

Doses / Concentrations:
620/598, 1242/1230, 2880/2550 mg/kg/day
Basis:
nominal in diet
females, mating/gestation

Remarks:

Doses / Concentrations:
2607, 5549, 10070 mg/kg/day
Basis:
nominal in diet
females, lactation

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

Food intake, clinical signs,

Litter observations:

Food intake, litter size, litter weight and the body weight of offspring
Sex ratio and survival index at PND 21

Functional and behavioral developmental parameters:
surface righting: PNDs 4 and 7
negative geotaxis: PNDs 4 and 7
cliff avoidance: PND 7
swimming behavior (direction, head angle, and limb movement): PNDs 4 and 14
olfactory orientation: PND 14

Exploratory behavior:
spontaneous motor activity: at 3 and 8 weeks of age
measurement parameters:number of movements, movement time (s), number of horizontal activities, total distance (cm), number of vertical activities, vertical time (s), number of turnings, average distance (cm), average speed (cm/s), and defecations

multiple water T-maze: at 7 weeks of age

Statistics:

Food intake, litter size, litter weight and the body weight of offspring were assessed with Bonferroni's multiple comparison test after analysis of variance (ANOVA), or the Kruskal-Wallis test. Sex ratio and survival index at PND 21 were assessed with the x2-test (2 x 2 or 2 x 4). The behavioral developmental data and movement activity data were assessed with the Mann-Whitney U-test (Martin and Bateson, 1990). Multiple water T-maze performance data was assessed with the signed Wilcoxon test for trials and assessed with the Mann-Whitney U-test among each treatment group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

increased food intake high dose

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

increased food intake high dose

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: increased intake high dose

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Food and chemical intake
There were few adverse effects of Allura Red AC on the average food intake during each period with the exception of the mating period. During the mating period, the average food intake was significantly increased in high-dosed group.
Therefore, the chemical intake was consistently increased as dose-related during each period.

F0 generation
Several dams showed under-development of mammary glands in the first week of lactation; one each in the control, low- and middle-dosed groups. One dam died during the second week of lactation in the low-dosed group.

Dose descriptor:

NOAEL

Effect level:

>= 2 550 - <= 10 070 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

reproductive performance

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

increased at lowe dose during lactation

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

F1 generation
No significant adverse effect was observed in either litter size or weight at birth. Sex ratio at birth was significantly reduced in the low-dosed group, while there were few adverse effects in other groups. The average body weight of offspring during the lactation period was significantly increased in the lower-dosed groups for each sex. Two litters died due to under-development of mammary glands of
their dams during the first week of lactation, one litter in the control and one in the low-dosed group, and one litter in the low-dosed group was destroyed humanely (to prevent them dying of starvation) in view of the death of their dam during the second week of lactation. The survival index at PND 21 showed no consistent adverse effect in treatment groups, while that for male offspring was significantly reduced in the low-dosed group and for female offspring was significantly increased in the high dose group.
As regards the neurobehavioral parameters, no adverse effect of Allura Red AC was found in the all parameters measured for behavioral development during the lactation period in each sex. Movement activity of exploratory behavior showed no adverse effect of Allura Red AC on pre-weaning period (at 3 weeks of age) for 5 min.
There were few adverse effects of Allura Red AC on multiple water T-maze performance in all treatment groups as compared with controls, while time taken was significantly reduced on the second trial in the low-dosed group of female and on the third trial in high-dosed group of both sexes as compared with the first trial. Movement activity of exploratory behavior showed no adverse effect of Allura Red AC on post-weaning period (at 8 weeks of age) for 10 min.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1.68 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

developmental neurotoxicity

Reproductive effects observed:

no

Conclusions:

From the results, the dose levels of Allura Red AC in the present study, which were at least 86-times greater (during the mating period in the low-dosed group), and up to 1430-times greater (during the lactation period in the high-dosed group), than the human ADI of 7.0 mg/kg body weight, produced few adverse effects on the reproductive and neurobehavioral parameters measured in mice. These results suggest that Allura Red AC will not produce adverse effects on reproduction and behavior at typical human intake levels.

Executive summary:

The color additive, Allura Red AC, was given in the diet to provide levels of 0.42, 0.84, and 1.68% (control, 0%), from 5 weeks of age of the F0 generation to 9 weeks of age of the F1 generation in mice, and selected reproductive and neurobehavioral parameters were measured. There were few adverse effects of Allura Red AC on either litter size or weight, and ratio of male to female was significantly reduced in the lowest dosed group. Average body weight of offspring during the lactation period was significantly increased in the lower dosed groups of each sex. As regards the neurobehavioral parameters, no adverse effect was observed in the behavioral development during lactation period. There were few adverse effects of Allura Red AC on either movement activity or maze learning in F1 generation mice, compared with controls in each sex. The dose levels of Allura Red AC in the present study (approximately 86–1430 times greater than human ADI) produced few adverse effects in reproductive and neurobehavioral parameters in mice.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 500 mg/kg bw/day

Species:

mouse

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication/study report which meets basic scientific principles

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Osborne-Mendel

Details on test animals or test system and environmental conditions:

Male and female Osborne Mendel (FDA strain) rats were obtained from the FDA rat breeding colony.
At the start of the study, female rats were 12-21 wk old and weighed 220-270 g.
Rats have been used traditionally in teratology studies, and the Osborne-Mendel FDA strain was chosen because of the availability of historical data.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

FD & C Red No. 40 was dissolved in distilled water (w/v) at concentrations of 3, 7.5, 15, 30, 60 and 100 mg/mL
Fresh solutions were prepared daily and administered by gavage in a volume of 1 ml/100 g body weight, at approximately the same
time each day.
Controls received an equivalent volume of distilled water.

Details on mating procedure:

Two females were randomly mated with one male
The following morning, a vaginal smear was obtained from each female to determine whether copulation had taken place.
Sperm-positive dams were considered to be at day 0 of gestation.

Duration of treatment / exposure:

Each animal was treated daily from day 0 to day 19 of gestation

Frequency of treatment:

daily

Duration of test:

until day 20 of gestation

No. of animals per sex per dose:

42-43 presumably pregnant females were assigned to each dose group by random number

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: gross abnormalities; Corpora lutea were counted. The uterus was opened and examined in situ. The uterine
positions of all implantation sites were noted and their condition (early or late resorptions, living or dead foetuses) was determined.

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Fetal examinations:

Each live foetus was promptly weighed, sexed and examined for gross external malformations, and the crown-rump length was measured. Any foetus that weighed less than 70% of the average weight of the concurrent male or female controls was considered to be a runt (Leuschner and Czok, 1973). Approximately one-half of the foetuses were fixed in alcohol, stained with Alizarin Red S and examined under a dissecting microscope for all skeletal variations. The remaining half of the foetuses were fixed in Bouin's solution, serially sectioned by razor blade and examined under a dissecting microscope for internal variations of the soft tissues.

Statistics:

All data analyses were performed by the Division of Mathematics at the FDA. Data on maternal initial body weights and food consumption were analysed by straight analysis of variance (ANOVA) and two-tailed t-test, and a regression analysis. The number of dams affected was analysed by a Fisher's exact test. Data on maternal weight gain were submitted to an analysis of covariance (ANOCOVA) and a two-tailed t-test. Data on
the numbers of implants, corpora lutea, total viable young and viable males and females were analysed by ANOVA followed by a one-tailed t-test. Data on implantation efficiency, early deaths, late deaths and total resorptions (early and late deaths) were transformed by using the Freeman-Tukey arc-sine transformation (Freeman and Tukey, 1950) and then analysed by ANOVA and a one-tailed t-test. Data on litters having one or more or two or more resorptions were analysed by a Fisher's exact test. Similar tests were applied to the number of runts per litter. Data on foetal body weights, crown-!o-rump lengths and foetal ossified vertebrae were analysed by nested ANOVA and a one-tailed t-test. The ANOVA included a correction for unequal sample size (Sokal and Rohlf, 1981). Data on the average number of foetuses per litter with skeletal, sternebral, combined
missing plus incomplete plus bipartite sternebrae or soft-tissue variations were transformed by using the Freeman-Tukey arc-sine transformation and then analysed by ANOVA and a one-tailed t-test. Litters with foetuses with at least one, at least two, etc.. skeletal, sternebral, combined missing plus incomplete plus bipartite sternebrae, or soft-tissue variations, and specific variations were analysed by Fisher's exact test.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No unusual behaviours were observed in the animals during the study. The external maternal findings were unremarkable. One female in the group given 300 mg/kg died at day 12 of gestation as a result of garage difficulties unrelated to dosage. The gavage difficulties, which occurred on day 3, caused the animal to consume only 4-5g food per day and consequently to lose weight during the last week of life.
Mean food consumption on days 0-7, 7-14, 14 20 and 0-20 by the treated animals was similar to that of the control animals. Initial body weight at day 0 and maternal body-weight gain during gestation were similar in all groups.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean foetal weights of males and females and crown rump lengths were similar in all groups.
No test substance-related skelettal or soft-tissue defects were noted.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

No dose-related maternal or developmental effects occurred when FD & C Red No. 40 was given by gavage at dose levels of up to 1 g/kg/day.The NOAEL for maternal developmental and teratogenic effects was 1000 mg/kg bw

Executive summary:

Osborne-Mendel rats were intubated with FD & C Red No. 40 at dose levels of 0, 30, 75, 150, 300, 600 or 1000 mg/kg body weight/day on days 0-19 of gestation. No developmental toxicity was observed when the animals were killed on day 20 of gestation. No dose-related changes were seen in maternal daily observations, food consumption, body-weight gain or implantations, or in foetal viability, body weight, body length, sex distribution or external variations. Skeletal and soft-tissue development appeared similar in foetuses of all groups. The isolated increases that occurred in the number of male foetuses, number of females with two or more resorptions, number of litters with three or more sternebral variations and incidence of 14th rib bud are considered random occurrences and were not related to dosage.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat