Calcium 4-[(5-chloro-4-methyl-2-sulphonatophenyl)azo]-3-hydroxy-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 48:2(Ca). In addition, fertility information of the structural analogues Pigment Red 57:1(Ca) and the sodium salt of Pigment Red 57 are available. Both Pigment Red 48:2(Ca) and 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 and 2009). In addition, no effects were observed in the one-generation fertility element of long-term feeding study with 0.05, 0.3 or 2% of the sodium salt of Pigment Red 57 (CTFA 1981).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Pigment Red 48:2
- CAS no. 7023-61-2
- Substance type: pigment
- Physical state: solid
- Analytical purity: 99%
- Date received: 2006-11-16
- Lot/batch No.: 061101
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Other: Identity confirmed by FT-IR

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan (Atsugi Breeding Center)
- Age at study initiation: 8 weeks
- Weight at study initiation: 326 - 376 g and 204 - 236 g
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.4 - 23.6°C
- Humidity (%): 49 -65 %
- Air changes (per hr): 6 - 20
- Photoperiod (hrs dark / hrs light): 07:00 - 19:00 light phase

Route of administration:

oral: gavage

Vehicle:

other: 1% Tween 80 in water

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): substance is a poorly soluble pigment
- Amount of vehicle (if gavage): 10 mL/kg

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.:

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

Dose / conc.:

200 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:

- Control: 7 males and 12 females plus each five recovery group animals
- 40 mg/kg bw: 12 males and 12 females
- 200 mg/kg bw: 12 males and 12 females
- 1000 mg/kg bw: 7 males and 12 females plus each 5 recovery group animals

Control animals:

yes, concurrent vehicle

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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males - day after final administration day
- Anaesthetic used for blood collection: Yes
- Animals fasted: No data
- For parameter see table
- How many animals: five per group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 43 and day 57
- Animals fasted: No data
- How many animals: five per group
- Parameters checked in table

URINALYSIS: Yes
- Time schedule: day 40 and day 54
- How many animals: five males per group
- Parameters: pH, protein, glucose, ketones, bilirubin, occult blood, urobilironen, urine colour

NEUROBEHAVIOURAL EXAMINATION: Function tests and motor activity performed for 5 animals (week 6)

Oestrous cyclicity (parental animals):

yes

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: not applicable

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:]

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- Organs investigated: heart, Lymph node, mandibular, Lymph node, mesenteric, Thymus, Spleen, Bone marrow, femur, Trachea, lung (and bronchus), Stomach, Small intestine - duodenum, Small intestine - jejunum, Small intestine - ileum, Large intestine - cecum, Large intestine - colon, Large intestine - rectum, Liver, Kidney, Urinary bladder, Testis, Epididymis, Seminal vesicle, Prostate, Coagulating gland, Pituitary, Thyroid, Parathyroid, Adrenal, Brain, Spinal cord, Sciatic nerve, Eyeball, Uterus, Ovary, Vagina

Histopathology evaluation was performed of the control and high dose group for all animals. If findings were observed, then also the slides of the lower dose groups and recovery animals were scored.

ORGAN WEIGHTS
Determination of organ weights (day 43 and day 57) Organ Weights: Thymus, Liver, Kidney, Testicles, Epididymis, Ovaries , Uterus, Brain, Heart

Postmortem examinations (offspring):

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

Statistics:

Multiple comparisons tests were performed with measured values : distribution uniformity was tested by Bartlett’s test , subsequently, one-way variance analysis was performed when the distribution was uniform, and Kruskal-Wallis’s test was performed when the homoscedastic was not recognized. Where significant differences were observed, Duneett’s test or Dunett’s type - multiple comparison tests were conducted.

Reproductive indices:

Copulation index, fertility index, implantation index, delivery index, gestation index

Offspring viability indices:

Litter size and viability index

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes attributed to the test substance

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes attributed to the test substance

Haematological findings:

no effects observed

Description (incidence and severity):

There were no changes attributed to the test substance

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no changes attributed to the test substance

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urine showed red coloration in some animals of the highest dose group on day 40, but not on day 54

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes attributed to the test substance

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- In the histological examination of animals sacrificed after the dosing period, degeneration/necrosis of the proximal tubular epithelium in the kidney was noted in males of the 1000 mg/kg group and females of the 40 mg/kg group and higher. Moreover, degeneration/necrosis of the papillary ductal epithelium in females of the 1000 mg/kg group and mild basophilic tubule in males of the 1000 mg/kg group and females of the 200 and 1000 mg/kg groups were noted.

- No abnormal changes in the digestive system or other organs/tissues in the histological examination were noted.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance-colored stool (red) was observed in all animals of the 40 mg/kg group and higher.

Reproductive performance:

no effects observed

The test substance had no effects on the reproductive parameters such as estrous cycle, mating index, fertility index, delivery index, gestation length, number of corpora lutea or implantations, implantation index, gestation index, or parturition or maternal behavior. On the examination of neonates, there were no significant differences in the number of offspring or live offspring, sex ratio, live birth index, or viability index on day 4. No abnormal findings attributed to the test substance were found in external features, clinical signs, body weights, or necropsy of the offspring.

Dose descriptor:

NOEL

Remarks:

fertility

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on fertility observed

Dose descriptor:

LOEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

There was no increase in the number of offspring with external anomalies or anury.

Dose descriptor:

NOEL

Remarks:

developmental toxicity

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 adverse developmental effects observed

Critical effects observed:

not specified

Reproductive effects observed:

no

Table 1: Histopathology findings in kidney - females (n = 5)

dose (mg/kg bw); R = recovery	0	40	200	1000	R0	R1000
Basophilic tubule grade 1	1	1	2	0	0	0
Basophilic tubule grade 2	0	0	1	1	0	0
Cell infiltration, inflammatory, focal, grade 1	1	0	0	0	0	0
Cell infiltration, inflammatory, pelvis, grade 1	0	0	0	0	1	0
Cyst	0	1	0	0	0	0
Degeneration/necrosis, papillary ductal epithelium, grade 1	0	0	0	1	0	0
Degeneration/necrosis, tubular epithelium, grade 1	0	1	1	0	0	0
Degeneration/necrosis, tubular epithelium, grade 2	0	0	2	2	0	0
Mineralization, medulla	0	0	0	0	2	1

Grade: 1 = Minimal; 2 = Mild; 3 = Moderate; 4 = Severe

Table 2: Histopathology findings in kidney - males (n = 5)

dose (mg/kg bw); R = recovery	0	40	200	1000	R0	R1000
Basophilic tubule grade 1	1	1	1	0	2	2
Basophilic tubule grade 2	0	0	0	4	0	0
Cell infiltration, inflammatory, focal, grade 1	0	0	0	1	0	0
Cyst	0	1	0	0	0	0
Degeneration/necrosis, tubular epithelium, grade 2	0	0	0	1	0	0
Dilatation, pelvis	0	1	0	0	0	0
Mineralization, medulla, grade 1	0	0	0	1	0	0
Mineralization, cortex, grade 1	0	1	1	1	0	0
Hyaline droplet, tubular epithelium, grade 1	0	0	0	0	0	1

Grade: 1 = Minimal; 2 = Mild; 3 = Moderate; 4 = Severe

 

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 48:2(Ca) was tested in a GLP-compliant screening study performed according to OECD testing guideline 422 at doses of 40, 200 and 1000 mg/kg bw. Pigment Red 48:2(Ca) had no effects on the reproductive parameters such as estrous cycle, mating index, fertility index, delivery index, gestation length, number of corpora lutea or implantations, implantation index, gestation index, or parturition or maternal behaviour. On the examination of neonates, there were no significant differences in the number of offspring or live offspring, sex ratio, live birth index, or viability index on day 4. No abnormal findings attributed to the test substance were found in external features, clinical signs, body weights, or necropsy of the offspring.

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 prenatal developmental toxicity studies are available for Pigment Red 48:2(Ca). Based on prenatal developmental studies performed with members of the same category and the OECD 422 study performed with Pigment Red 48:2, it can be concluded that Pigment Red 48:2(Ca) is not a developmental toxicant.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Please see the category read-across justification in the category object.

Reason / purpose for cross-reference:

read-across source

Species:

rat

Dose descriptor:

NOAEL

Remarks:

Developmental / teratogenicity

Effect level:

> 50 mg/kg bw/day

Based on:

other: read-across

Basis for effect level:

other: no adverse effects expected

Abnormalities:

no effects observed

Dose descriptor:

NOAEL

Effect level:

> 50 mg/kg bw/day

Based on:

other: read-across

Sex:

male/female

Basis for effect level:

other: no adverse effects expected

Abnormalities:

no effects observed

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 with Pigment Red 57:1(Ca) 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) and 48:2(Ca) (MHLW 1993 and 2009).

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.