Sodium [4-[[dihydroxy[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]benzenesulphonato(3-)]cuprate(1-)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 16, 2016 - Jan 11, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study done to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium [4-[[dihydroxy[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]benzenesulphonato(3-)]cuprate(1-)

EC Number:

305-729-0

EC Name:

Sodium [4-[[dihydroxy[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]benzenesulphonato(3-)]cuprate(1-)

Cas Number:

95009-01-1

Molecular formula:

C18H9CuN6O10S.Na

IUPAC Name:

Reaction products of diazotized 4-aminobenzenesulfonic acid coupled with resorcinol subsequently coupled with diazotised 2-amino-4,6-dinitrophenol, copper (2+) complexes, sodium salts

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Everlan SL65
- Substance type: Powder
- Composition of test material, percentage of components: 71.19%
- Lot/batch No.: 3010
- Storage condition of test material: Ambient

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: BioLASCO Taiwan Co., Ltd., Taipei, Taiwan
- Age at study initiation: about 9-week old
- Weight at study initiation: Males: 330-383 g; Females: 211-253 g
- Housing: Except for the mating period, animals were housed individually in polycarbonate cages. While mating, animals were pair-housed in stainless steel wire mesh cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 50 ± 20%
- Photoperiod: 12-hrs dark / 12-hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Water for injection

Details on mating procedure:

- M/F ratio per cage: 1:1
- After 4 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): 1 animal per cage

Duration of treatment / exposure:

For male: 28 days
For female: 40-53 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Dose Group 1 (Control)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Dose Group 2 (low dose)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Dose Group 3 (mid dose)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Dose Group 4 (high dose)

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

These observations included red-brown colored feces, soft feces, unformed feces, amber/red-brown/yellow colored urine, the red, red-brown, yellow or brown hair stains on the whole body and red hair stains on the nose in all groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males, statistically significantly reduced body weight gain compared to the controls was observed in Group 4 animals on Day8. The total body weight gain of Group 4 was also lower than the body weight gain of the control animals between D1 and D 29, but the difference did not reach statistical significance difference.
In females, reduced body weight gain was also observed reaching statistical significance compared to the controls in Group 4 on D15 and in Groups 3 and 4 on G6.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly increased food consumption compared to the controls was observed in males of Groups 3 and 4 on D15, in females of Group 2 on D13 and in females of Group 4 on G6, G13, G20 and P4.

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Discolouration of urine from Day 2 in high dose groupe rats.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences in the number of corpora lutea and implantations between control and treated groups. Pre-implantation and pre-natal losses were observed in all groups including the controls. The only treatment-related gross finding was discoloration of the cecum in 3 animals of Group 4. Decreased size of thymus was observed in one animal each of Groups 3 and 4

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

In males, a positive indication of mating was obtained for 9, 10, 9 and 8 of 10 animals in Groups 1 to 4, resulting in a mating index of 90%, 100%, 90% and 80%, respectively, indicative of a slight decrease at Group 4.
In females, a positive indication of mating was obtained for 10 of 10 animals in Groups 1, 2 and 3 and for 9 of 10 animals in Group 4. The mating index was 100%, 100%, 100% and 90% for Group 1 to 4, respectively.
There was one animal in Group 1 and 3, respectively, that showed a pre-coital interval longer 5 days. In each of these females, the mating with the first male failed but the copulation with another male was successful. In Groups 2 to 4, 1 or 2 animals with positive indications of mating was later found not to be pregnant. All Group 1 animals were found to be pregnant. Thus, the fertility index was 100%, 90%, 90% and 78% in the control, low, mid and high dose group, respectively, indicative of a slight decrease in Group 4. The average length of gestation was 21.7-22 days without dose-dependency.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

All pregnant rats delivered live pups. The gestation index was 100% in all groups. One and three dead pups were observed at birth in Groups 2 and 4. The incidence of dams with dead pups at birth was 0%, 11%, 0% and 29% in Group 1, 2, 3 and 4, respectively. The dead pup incidence in Group 4 was slightly higher than in other groups. However, the number of pups per litter at birth in Group 4 was higher than in other group. Moreover, the number of stillborns per litter was 0.42. On P4, a decreased number of live pups was observed in Groups 2 and 4. The 4-day survival index was 100.0%, 97.0%, 100% and 97.2% in Groups 1 to 4, respectively. No external abnormally pups were observed at birth in the control and treated groups. At birth, 45.4% to 54.2% of pups were male and on P4, 46.2% to 59.6% pups were male. The litter weights in all groups increased from P0 to P4.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food efficiency

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

1

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

testes

2

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

uterus

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

other:

Remarks on result:

not measured/tested

Remarks:

OECD 421 method is not defined the does object for the pups.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Any other information on results incl. tables

For male: DX mean DayX

For female: DX mean DayX during pre-mating; GX mean DayX during gestation; PX mean DayX during lactating

 

Dose Formulation Analysis

On the first and last preparation, the difference between the mean value and the targeted concentration for all dose group formulations were within ± 15.0%. The homogeneity verification revealed that the precision of samples for the low and high dose formulation were ≤ 10.0%.

 

Mortality and Clinical Observations

All animals survived the study period. These observations included red-brown colored feces, soft feces, unformed feces, amber/red-brown/yellow colored urine, partial hair loss, the red, red-brown, yellow or brown hair stains on the whole body and red hair stains on the nose in all groups. For only one male and one female rat at the high dose level, the salivation was observed for two days.

 

Body Weights

In males, statistically significantly reduced body weight gain compared to the controls was observed in Group 4 animals on Day8. The total body weight gain of Group 4 was also lower than the body weight gain of the control animals between D1 and D 29, but the difference did not reach statistical significance difference. In females, reduced body weight gain was also observed reaching statistical significance compared to the controls in Group 4 on D15 and in Groups 3 and 4 on G6.

 

Food Consumption

Statistically significantly increased food consumption compared to the controls was observed in males of Groups 3 and 4 on D15, in females of Group 2 on D13 and in females of Group 4 on G6, G13, G20 and P4.

 

Cohabitation and Pregnancy

In males, a positive indication of mating was obtained for 9, 10, 9 and 8 of 10 animals in Groups 1 to 4, resulting in a mating index of 90%, 100%, 90% and 80%, respectively, indicative of a slight decrease at Group 4. In females, a positive indication of mating was obtained for 10 of 10 animals in Groups 1, 2 and 3 and for 9 of 10 animals in Group 4. The mating index was 100%, 100%, 100% and 90% for Group 1 to 4, respectively. There was one animal in Group 1 and 3, respectively, that showed a pre-coital interval longer 5 days. In each of these females, the mating with the first male failed but the copulation with another male was successful. In Groups 2 to 4, 1 or 2 animals with positive indications of mating was later found not to be pregnant. All Group 1 animals were found to be pregnant. Thus, the fertility index was 100%, 90%, 90% and 78% in the control, low, mid and high dose group, respectively, indicative of a slight decrease in Group 4. The average length of gestation was 21.7-22 days without dose-dependency.

 

Litter Observation

All pregnant rats delivered live pups. The gestation index was 100% in all groups. One and three dead pups were observed at birth in Groups 2 and 4. The incidence of dams with dead pups at birth was 0%, 11%, 0% and 29% in Group 1, 2, 3 and 4, respectively. The dead pup incidence in Group 4 was slightly higher than in other groups. However, the number of pups per litter at birth in Group 4 was higher than in other group. Moreover, the number of stillborns per litter was 0.42. On P4, a decreased number of live pups was observed in Groups 2 and 4. The 4-day survival index was 100.0%, 97.0%, 100% and 97.2% in Groups 1 to 4, respectively. No external abnormally pups were observed at birth in the control and treated groups. At birth, 45.4% to 54.2% of pups were male and on P4, 46.2% to 59.6% pups were male. The litter weights in all groups increased from P0 to P4.

 

Male Gross Examination, Organ Weight and Histopathology

There were no statistically significant differences in testis and epididymis weights between control and treated groups. No gross changes were observed in any males.

 

Females Gross, Uterus Examination and Histopathology

There were no statistically significant differences in the number of corpora lutea and implantations between control and treated groups. Pre-implantation and pre-natal losses were observed in all groups including the controls. The only treatment-related gross finding was discoloration of the cecum in 3 animals of Group 4. Decreased size of thymus was observed in one animal each of Groups 3 and 4

Applicant's summary and conclusion

Conclusions:

According to OECD 421 test method, the no observed adverse effect level of Everlan SL65 in rat's reproductive performance was 1000 mg/kg/day.

Executive summary:

This test using the procedures outlined in the QPS Study Plan for T65315036-RP and OECD 421. Everlan SL65 (100, 300 or 1000 mg/kg/day) was given orally by gavage once daily to male rats for 28 days and female rats for 40 to 53 days, depending on the time of copulation and gestation. Dose-related clinical observations in the treated groups included test article-like colored/soft/unformed feces or urine and hair stained. Reduced body weight gain and increased food consumption were observed in males and females at Group 3 or 4. The only treatment-related gross findings was discoloration of the cecum in three females at Group 4. However, there were no definitely test article-related effects on male and female rats’ reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition. The no observed adverse effect level of Everlan SL65 in rat’s reproductive performance was 1000 mg/kg/day.