Calcium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Experimental data regarding fertility is available for Pigment Red 57:1(Ca). Pigment Red 57:1(Ca) caused no effects on fertility in a GLP-compliant screening study performed with up to 1000 mg/kg bw according to OECD testing guideline 422 (MHLW 1993).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Combined Repeated Dose and Reproductive / Developmental Toxicity Screening Test (Precursor Protocol of GL 422)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

-Analytical purity : 98 % w/w
-Lot/batch No.: T-1322-2
-Appearance : Red powder

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Sources:Charles River Laboratories Japan (Atsugi Breeding Center)
- Age at study initiation : 7 weeks
- Weight at study initiation : females 196.5-227.4 g, males: 235.5-299.6 g
- Housing : Individually housed in stainlelss suspended cages
- Diet : CA-1 (CLEA Japan, Inc.) ad libitum
- Water : Tap water, ad libitum
- Acclimation period : 1 week for quarantine and acclimaition

Route of administration:

oral: gavage

Vehicle:

other: 5% gum arabic solution

Details on mating procedure:

- M/F ratio per cage: 1:1

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males: Total of 42 days ( 14 days before mating, 14 days for mating, and 14 days after mating)
Females Total of 41-50 days (14 days before mating, during mating, pregnancy and up to lactation day 3)

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

13/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected on the basis of a 14-day preliminary repeat dose study at 1000 mg/kg bw/day. No deaths and clinical signs observed. 3 cases of necrosis or regeneration of tubular epithelium were seen in males.

Positive control:

not used

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males and Females - on each administration day.

BODY WEIGHT: Yes
- Time schedule for examinations: Males - measured once a week on administration days 0, 7, 14, 21, 28, 35, and 42; Females - measured once a week on administration days 0, 7, 14, and 21, during pregnancy on days 0, 7, 14, and 20, during lactation on day 0 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined: Yes
- Time schedule for examinations : Males - measured once a week on administration days 0, 7, 14, 21, 28, 35, and 42; Females - measured once a week on administration days 0, 7, 14, and 21, during pregnancy on days 0, 7, 14, and 20, during lactation on day 0 and 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males - day after final administration day
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: No data
- How many animals: 13 animals per male per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Males - day after final administration day
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: No data
- How many animals : 13 animals per male per group
- Parameters checked in table 2 were examined.

Litter observations:

STANDARDISATION OF LITTERS
not applicable


PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals


GROSS NECROPSY
- yes, see entry for repeated dose toxicity for details


HISTOPATHOLOGY / ORGAN WEIGHTS
- yes, see entry for repeated dose toxicity for details

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

x squared test was conducted for copulation and fertility indices. For all the other parameters, Bartlett's test was used to examine uniformity of distribution in each group, and one-way statistical analysis was performed when the distribution is uniform. Where significant differences were observed, Duneett's test or Scheff's test was performed to examine differences in averages between each test group and control groups. Kruskal-Wallis's test was conducted when the distribution was not uniform. Significant testing was carried out at the 5% and the 1% levels.

Reproductive indices:

yes

Offspring viability indices:

yes

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males - 1 day after termination of administration period: Significantly lower values of MCH (p<0.05) were seen in the 300 and 1000 mg/kg bw/day groups compared to the controls. Dose-dependent decrease in WBC was seen in the the 300 and 1000 mg/kg bw/day groups, but the effects were not significant. In the absence of historical control data, no corresponding histopathology findings and other affected parameters, the findings are considered of no biological relevance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males - 1 day after termination of administration period: Significantly lower levels of inorganic phosphorus and Calcium were seen in the 300 mg/kg bw/day group. Significant low levels of Total cholesterol and pottasium and significant high values of Chloride and GOT were noted in the 1000 mg/kg bw/day group.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MALES - 1 day after termination of administration period:
Thymus: Two cases of hemorrhage were seen in the 1000 mg/kg bw/day group, one case in the control group. In the absence of a dose-response relationship, this is considered incidental.
Heart: Small areas of myocardial degeneration were seen in 2 cases in the control group. No other abnormalities were observed.
Liver: No treatment-related findings were observed. Ten cases of very slight microgranuloma were seen respectively in the control and the 1000 mg/kg bw/day groups. Slight fatty changes in peripheral zone were seen in a case of the control group and in four cases of the 1000 mg/kg bw/day group. Therefore, there was no significant difference in severity and incidence of the findings between the control and the 1000 mg/kg bw/day groups. A case of very slight focal necrosis was seen in the 1000 mg/kg bw/day group.
Kidney: Large numbers of regenerating tubular epithelium were observed in three cases in the 300 mg/kg bw/day group and twelve cases in the 1000 mg/kg bw/day group compared to the control groups. The severity was augmented in the 1000 mg/kg bw/day group. The regenerating epithelial cells were found predominantly in convoluted proximal tubules, showing increased cell density, slightly enlarged nucleoli, and slightly bright or basophilic cytoplasm. In most cases, slightly-yellowish debris was noted in the tubular lumen. A single case of cast in tubular lumen was found in the 100 mg/kg bw/day group. In all groups, including the control group, eosinophilic bodies were observed with no significant differences in incidence and severity among the groups.
Adrenal cortex : Brown pigment deposits were observed both in the 1000 mg/kg bw/day and the control groups but there is no significant difference in incidence and severity.
Spleen: Brown pigment deposits and extramedullary hematopoiesis were found both in the 1000 mg/kg bw/day and the control groups, with no significant difference in incidence and severity.
Testis: Atrophy of tubule was found in 2 cases in the control group and 3 cases in the 1000 mg/kg bw/day group. One of the cases in the 1000 mg/kg bw/day group had calcification in the tubule.
Epididymis: Decreased numbers of sperm were noted in each one case in the control and the 1000 mg/kg bw/day groups. Both animals had atrophy of the tubule.

FEMALES
Thymus: A case of very slight and a case of slight involution were found in the control group. In the 1000 mg/kg bw/day group, increased numbers of involution were found with a case rated "very slight", 3 cases rated "slight", and 2 cases rated "moderately slight".
Liver: Very slight microgranuloma was seen in a case of the control and 2 cases of the 1000 mg/kg bw/day group sacrificed at Day 4 of lactation, and one non-pregnant animal in the control group sacrificed at Day 25 of pregnancy. Very slight to slight fatty changes in peripheral zone were seen in 3 cases in the control group, and very slight to moderate fatty changes in 2 cases in the 1000 mg/kg bw/day group. There were no significant differences in severity and incidence between the control and the 1000 mg/kg bw/day groups.
Kidney: In all treatment groups sacrificed at Day 4 of lactation, increased numbers of incidences of regenerating tubular epithelium were observed. The findings are accompanied with foamy epithelial cells and vacuolar degeneration predominantly in convoluted proximal tubule. In many cases, necrotized epithelial cells were noted, and eosinophilic necrotized cells and yellowish debris were contained in the tubular lumen. In tubular basement of these animals, regenerating epithelial cells included large-sized basophilic cytoplasma. The incidence was similar in all dose groups; the severity of this lesion was slightly increased in the high-dose group.
In all dose groups, there were some animals completely free of kidney findings. Among those were a non-delivering animal (total implantation loss) in the 100 mg/kg bw/day group, 2 non-pregmant animals in the 300 mg/kg bw/day group, and a non-copulated animal in the 1000 mg/kg bw/day group.

Other kidney findings that are considered incidental included each one case of cast in the tubular lumen in the control and the 1000 mg/kg bw/day groups, each one case of slight vacuolar degeneration in the control and the 100 mg/kg bw/day groups, and each one case of focal dilatation of tubule in the 100 mg/kg bw/day and the 1000 mg/kg bw/day groups. The case of cast of the animal in the control group was considered be associated with the very slight incidence of chronic nephropathy.

Adrenal cortex: A case of very slight brown pigment deposit and a case of focal necrosis were observed in the 1000 mg/kg bw/day group.
Spleen: Brown pigment deposits and extramedullary hematopoiesis were observed in 11 animals in the control group and 12 animals in the 1000 mg/kg bw/day group. Two cases were also found in non-pregnant animals in the control group, sacrificed at Day 25 of pregnancy. There were no clear differences in incidence and severity between the treated and the control groups.
Ovary: No abnomarities were found with non-pregnant animals (two in the control and two in the 300 mg/kg bw/day groups) and in the non-copulated animal (one in the 1000 mg/kg bw/day group).

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOEL

Effect level:

< 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Adverse effects on kidneys of parental animals observed at all dose groups.

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

No treatment-related adverse effects on the offspring was observed.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on offspring observed

Critical effects observed:

no

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Pigment Red 57:1(Ca) caused no effects on fertility in a valid screening study performed according to OECD testing guideline 422 at doses of 100, 300, and 1000 mg/kg bw/day (MHLW 1993). Up to the highest dose of 1000 mg/kg bw, no indication on female or male fertility were observed. The 14-day pre-mating period allows only a limited assessment of male fertility, but further information on male fertility can be derived from the fertility element and the histopathology of the long term feeding study with the sodium salt of Pigment Red 57. The fertility element is similar to the one-generation study (OECD 415) with the limitation that 60 instead of 70 days treatment prior to mating are used. No adverse effects on fertility or reproductive organs were observed at dose levels in the diet of up to 2%. This study is adequate in design and reporting for hazard assessment. Discoloration of urine by the test item indicates systemic availability despite the better water solubility compared to Pigment Red 57:1(Ca).

Results regarding a three-generation study with Pigment Red 57:1(Ca) in rats are mentioned in various secondary sources. The actual study (Weil 1973) was performed following a non-standard design with doses of 0.5, 5, 15 and 50 mg/kg bw. Rats are treated via the feed for three generations and each generation produced one to three sets of offspring which were used for mating or pathology. Historical control data was not available. Analysis in the feed was not performed. Limited details are reported regarding macroscopic and microscopic examinations. The study reports effects on fertility for males of the second generation at 50 mg/kg bw, but not of the first or third generation. Histopathology investigations on reproductive organs were apparently not performed for the F2 generation. Detailed data is only given for the three control groups and the high dose group of the F1b group regarding lung, liver, kidney, heart, adrenal, thyroid, trachea and prostate. The findings of the three-generation study are poorly reported and inconclusive and therefore do not contribute to hazard assessment. No effects on lactation were observed.

Effects on developmental toxicity

Description of key information

No teratogenicity of Pigment Red 57:1(Ca) was found in a study in rats with 50 mg/kg bw (Durloo 1972). In addition, no developmental toxicity was observed in a GLP-compliant screening study performed with up to 1000 mg/kg bw according to OECD testing guideline 422 (MHLW 1993 and 2009) with Pigment Red 57:1(Ca), and the structural analogue Pigment Red 48:2(Ca).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1972

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Little reporting details on animals, test item and results. Highest dose is only 50 mg/kg bw and no maternal toxicity mentioned.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

low dose, limited investigations regarding maternal toxicity. No individual data, no statistical evaluation

GLP compliance:

no

Remarks:

pre-dates GLP requirements

Limit test:

no

Specific details on test material used for the study:

D&C Red No. 7 (FDA Certification no. Y W4502)

Species:

rat

Strain:

other: CD

Details on test animals or test system and environmental conditions:

Male and female CD rats were obtained from Tthe Charles River Breeding Laboratory an weanlings, the males assigned numerical identification at random and placed in individual hanging cages until they reached a satisfactory age for mating. The diet consisted of Puriria Laboratory Chow. Body weight of dams of gestation ranged between 213 and 292 g on day 0.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose

Details on exposure:

Volumes of 10, 3-2, and 1 mL/kg were used with all controls receiving a volume of 10 mL/kg.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Mating was accomplished by pairing one female with one male. The appearance of a vaginal plug was taken as evidence of satisfactory mating and this was counted as day zero of gestation.

Duration of treatment / exposure:

day 6 - 15 of pregnancy

Frequency of treatment:

daily

Duration of test:

Dams wew sacrificied on day 20 of gestation ander chloroform anaesthesia.

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Dose / conc.:

16 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

- Control: two groups, each with 22 females
- Test group: 22 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Acetylsalicylic acid (aspirin, 250 mg/kg bw) was used as positive control.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 15 and 20 of gestation


POST-MORTEM EXAMINATIONS: No
- Sacrifice on gestation day 20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No, but total litter weight determined
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of resorptions: Yes (no difference made for early or late)
- Other: number of dead and live fetuses were recorded, sex distribution was recorded.

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes: one third per litter
- Skeletal examinations: Yes: two thirds per litter
- Head examinations: No data
- Fetal weight: determined per litter litter

Statistics:

none

Indices:

none

Historical control data:

The results were compared to historical control data, but no actual historical control data is included in the report.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Groups of ten females given 5, 16 or 50 mg/kg bw/day by stomach tube on days 6-15 of pregnancy showed no adverse effects on maternal weight gain.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 50 mg/kg bw/day

Basis for effect level:

other: no adverse effects observed

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Groups of ten females given 5, 16 or 50 mg/kg bw/day by stomach tube on days 6-15 of pregnancy showed no adverse effects on number of resorptions, litter size, foetal weight and viability, or the incidence of foetal malformations. Animals of the positive control group (aspirin at 250 mg/kg bw) showed the expected malformations.

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

>= 50 mg/kg bw/day

Basis for effect level:

other: no adverse effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Study reports are available for two teratogenicity studies in rats and in rabbits (Durloo 1972). The study in rabbits is of limited value as only 10 pregnant females per dose group were used. Pregnant rabbits received 5, 16 or 50 mg/kg bw by gavage during gestation days 6 - 18 and they were sacrificed on gestation day 29. No effects were recorded regarding the number of viable and dead fetuses, resorption sites, mean fetal weight, distribution of sex, mean litter size, frequency of skeletal anomalies and frequency of visceral and structural anomalies.

The study in rats was performed with doses of 5, 16 and 50 mg/kg bw, applied by gavage during gestation days 5 to 15. No adverse effects were reported regarding the same endpoints as for rabbits. The study included limited investigations regarding maternal toxicity, as only body weight gain of dams was reported. No individual data and no statistical evaluation are given in the report. The highest dose is slightly above the subacute NOAEL for adverse effects on kidney.

Furthermore, no indication of developmental toxicity was observed in the OECD 422 screening studies with Pigment Red 57:1(Ca)(MHLW 1993).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for reproductive toxicity according to Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008.