Calcium 4-[(5-chloro-4-methyl-2-sulphonatophenyl)azo]-3-hydroxy-2-naphthoate

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Calcium 4-[(5-chloro-4-methyl-2-sulphonatophenyl)azo]-3-hydroxy-2-naphthoate

EC Number:

230-303-5

EC Name:

Calcium 4-[(5-chloro-4-methyl-2-sulphonatophenyl)azo]-3-hydroxy-2-naphthoate

Cas Number:

7023-61-2

Molecular formula:

C18H13ClN2O6S.Ca

IUPAC Name:

calcium 4-[(5-chloro-4-methyl-2-sulfonatophenyl)diazenyl]-3-hydroxy-2-naphthoate

Test material form:

solid: nanoform

Details on test material:

- 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%
- Impurities: No data
- Lot/batch No.: 061101
- Stability under test conditions: Stable, no change before and after the administration period. (confirmed by IR analysis)
- Storage condition of test material: Stored in a sealed container, at room temperature ( Measured temperature: 16.2-26.4 °C, Acceptance range: 10 -30 oC), in a dark place.

Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 function / performance (P0)

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

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

2

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

3

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

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Results: F1 generation

Details on results (F1)

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

Effect levels (F1)

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

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

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