Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(1-sulphonato-2-naphthyl)azo]naphthalene-2,7-disulphonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Mar - 15 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See chapter 13 for detailed read across justification.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

23 Mar - 15 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See chapter 13 for detailed read across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted in 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Limit test:

no

Species:

rat

Strain:

other: Wistar Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: (P) 10-11 wks
- Weight at study initiation: (P) Males: 257-304 g; Females: 175-213 g
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (Lot. No. 081110)
- Diet: Altromin 1324 maintenance diet for rats and mice (Lot. No. 1307), ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark/hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The amount of test substance for each dose concentration was suspended separately in aqua ad injectionem (sterile water) on each administration day, immediately before the administration. Homogeneity was ensured by using a vortex machine.

VEHICLE
- Justification for use and choice of vehicle: Selection was based on the solubility of the test item.
- Concentration in vehicle: 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no.: 0195A191

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- Females with unsuccessful mating were allowed to mate with other male of the same group.
- Females showing no evidence of copulation up to 14 day mating period were sacrificed 26 days after the last day of the mating period.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each concentration was analysed for nominal concentration. Homogeneity in the vehicle was analysed for the low and high dose concentrations. Samples for nominal concentration verification were taken in Week 1 (first week of pre-mating period), Week 3 (first week of mating), Week 5 (gestation) and Week 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the low and high dose preparation in Weeks 1 and 5.
All concentration samples were stored frozen (approx. -20 °C) until the analysis was performed.

Duration of treatment / exposure:

Males: 14 days before mating, 14 days during mating
Females: 14 days before mating, 14 days during mating, 20 days during gestation, 3 days during lactation

Frequency of treatment:

daily

Details on study schedule:

- Age at mating of the mated animals in the study: 12-13 weeks

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

other: yes, concurrent vehicle; the control group was shared with BSL Study no. 110998, another OECD 421 study, which was performed in parallel.

Details on study design:

- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. A descending sequence of dose levels was selected in order to demonstrate any dose-related response and a NOAEL.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily during weekend/holidays

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before assignment to the experimental groups and on the first day of administration.
Males were weighed weekly during the entire study period.
Females were weighed weekly during the pre-mating period, on Gestation Day 0, 7, 14, 20 and on Post-natal Day 0 (within 24 hours of parturition) and Post-natal Day 4 along with pups.

FOOD CONSUMPTION:
- Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration and was not measured during the mating period.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after the completion of mating period (Day 28)
- Maternal animals: All surviving animals were sacrificed on respective PND 4.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively:
ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), macroscopic lesions

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspringwere sacrificed at 4 days of age (PND 4).

The animals were subjected to postmortem examinations for gross external abnormalities.

Statistics:

One-way analysis of variance (ANOVA) followed by DUNETT’s multiple comparison test (p<0.05 was considered as statistical significant).

Reproductive indices:

Copulation Index (%) = (No. of rats copulated /No. of pairs) x 100
Fertility Index (%) = (No. of females pregant/No. of females copulated) x 100
Delivery Index(%) = (No. of dams with live newborns/ No. of pregnant dams) x 100

Offspring viability indices:

Viability Index (%) = (No. of live offspring at Day 4/ No. of live offspring at birth) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

histopathology correlates to discoloration in organs (see details below)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No treatment related clinical signs were observed. There were very few clinical signs recorded in control and treated animals during the study period but these findings were observed transiently post dose administration and had no relevance to treatment.
None of the animals died during the study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No effect on body weight was observed in both sexes throughout the complete study period.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS):
No treatment-related effects on testis or epididymidis weight were noted. No other sperm parameters were examined.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No treatment related effect on precoital interval and duration of gestation was observed.
The copulation index, fertility index and delivery index remained unaffected. No effects on mean No. of corporea lutea, No. of implantation sites, No. of live pups born, % pre- and post-implantation loss were observed.

ORGAN WEIGHTS (PARENTAL ANIMALS):
No statistically significant changes in absolute and relative organ weights were observed.

GROSS PATHOLOGY (PARENTAL ANIMALS):
In males, various macroscopic findings observed were yellow spots on epididymides (C:1/10, LD:1/10, MD:1/10), discoloration of epididymides (HD:5/10 animals), reddish discoloration of kidneys (LD: 4/10, MD: 6/10, HD: 10/10 animals), reddish discoloration of testes (HD: 3/10 animals), small testes and epididymides (HD: 1/10 animals).
In females, discoloration of kidneys (LD: 7/10, MD: 6/10, HD: 10/10 animals), cyst on kidneys (LD: 1/10 animal), ureter dilated (HD: 1/10 animal), dark discoloration of lung (HD: 1/10 animal), hardening of ovary (HD: 1/10 animal).
The discoloration of organs as well as reddish discoloration of the digestive tract noted in almost all treated animals is considered to be attributable to the colour of the test item and as such not a systemic effect due to the test item administration.

HISTOPATHOLOGY (PARENTAL ANIMALS):
There were foamy interstitial macrophages observed in testes of one male of MD group and in 9/10 males of HD group. There were no test item-related histological findings noted in other male and female reproductive organs.
Eosinophilic granules in the tubular epithelium of the renal cortex were seen in all animals of HD group and in a proportion of animals of MD group, but not in animals of LD group although macroscopically red discolored. This finding was considered to be due to test item deposition in renal tubuloepithelial cells. Moreover, in all females of the high dose group, renal corticotubular epithelium was diffusely vacuolated, possibly caused by washout of the test item during the histotechnical procedure.
The lung of one high dose female, being macroscopically dark discolored, showed minimal numbers of eosinophilic alveolar macrophages, considered to represent test item deposition.
All histological findings are due to test item deposition and there were no accompanying findings that would indicate organ damage.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects noted.

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects noted.

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING):
No treatment related effect was observed on total No. of pups born, No. of males, No. of females, sex ratio, live pups, still birth and runt on PND 0 and total No. of live pups and sex ratio on PND 4. Viability index was unaffected.

BODY WEIGHT (OFFSPRING):
No effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4.

GROSS PATHOLOGY (OFFSPRING):
No substance-related gross external findings were observed in any of the treated groups.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects noted.

Reproductive effects observed:

not specified

Table 1: Clinical Observations (P)

Clinical Findings	Control	100 mg/kg	300 mg/kg	1000 mg/kg
Males
Aggressive behavior	0/10	0/10	3/10	0/10
Nasal discharge	1/10	0/10	0/10	0/10
Piloerection	0/10	0/10	0/10	1/10
Salivation	0/10	0/10	1/10	0/10
Alopecia	1/10	0/10	1/10	0/10
Females
Aggressive behavior	1/10	0/10	0/10	0/10
Nasal discharge	0/10	2/10	0/10	0/10
Piloerection	0/10	1/10	0/10	1/10
Alopecia	0/10	1/10	2/10	2/10

 

Table 2: Macroscopic Findings (P)

Findings	Control	100 mg/kg	300 mg/kg	1000 mg/kg
Males
Epididymides: yellow spots	1/10	1/10	1/10	0/10
Epididymides: discoloration (reddish)	0/10	0/10	0/10	5/10
Epididymides: small	0/10	0/10	0/10	1/10
Kidney: discoloration (reddish)	0/10	4/10	6/10	10/10
Testes: discoloration (reddish)	0/10	0/10	0/10	3/10
Testes: small	0/10	0/10	0/10	1/10
Females
Kidney: discoloration (reddish)	0/10	7/10	6/10	10/10
Ureters: dilated	0/10	0/10	0/10	1/10
Lung: discoloration (dark)	0/10	0/10	0/10	1/10
Ovary: hard	0/10	0/10	0/10	1/10

 

Table 3: Reproductive Indices (%)

	Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Copulation Index	100	100	100	100
Fertility Index	60	80	70	60
Delivery Index	100	100	100	100
Viability Index	100	100	100	100

Conclusions:

The test substance had no effect on reproductive performance.
Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40850/B TE, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg body weight for reproduction toxicity screening in males and females.
CLP: not classified
DSD: not classified

Executive summary:

Currently no study to assess the reproductive and/or developmental toxicity of Reactive Red 218 is available. Nevertheless, data from a Read across substance FAT 40850 is available. FAT 40850 was evaluated in a study performed according to OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

In this study, four groups comprised of 10 adult male and 10 non pregnant nulliparous female rats [Wistar Crl:WI(Han)] were dosed daily by oral gavage with 100, 300 and 1000 mg/kg body weight per day of FAT 40850/B TE at dose volume of 10 mL/kg body weight. The test item was formulated in sterile water with an administration volume of 10 mL/kg body weight. Control animals were handled identically as treated groups and received sterile water in similar volume as treated groups.

 

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period. After 14 days of treatment to both male and female, animals were paired (1:1) for maximum 14 days. The subsequent morning onwards, vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. Males and females were sacrificed on day 29 and post natal day 4 respectively and subjected to necropsy. Non pregnant females were sacrificed on their respective day 26 after the evidence of mating or completion of mating period.

 

There were no predominant clinical signs considered to be due to treatment observed in treatment groups compared to the controls. There were no mortalities observed in males or females during the study period. In males and females, statistical analysis of body weight data revealed no effect on body weight throughout the study period in treated groups when compared with controls.

 

There was no treatment related effect observed for duration of gestation and precoital interval in treated groups when compared with controls. All females in control and treated groups showed evidence of copulation during 14 days mating

period. Successful mating of females resulted in 60 % pregnancy rate each in the control and HD group, while 80% and 70% pregnancy rate in LD and MD groups, respectively.

The copulation index, fertility index and viability index in treated groups remained unaffected due to treatment when compared to the control. All pregnancies resulted in normal births and therefore delivery index remained unaffected in all treated groups. There was no treatment related effect observed on total number of pups born, number of male pups, number of female pups, sex ratio, live pups, still birth and runt on PND 0 and total number of live pups and sex ratio on PND 4 in treated groups compared to corresponding control. Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treated groups compared to the control.

 

In males and females, there were no treatment related changes observed in the absolute and relative organ weights of the treatment groups when compared with the controls. Histopathological evaluation showed there was no histomorphological indication of any functional impairment of the organs and tissues concerned.

 

Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40850/B TE, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg body weight for reproduction toxicity screening in males and females.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted in 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

FAT 40850/B TE

IUPAC Name:

FAT 40850/B TE

Test animals

Species:

rat

Strain:

other: Wistar Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: (P) 10-11 wks
- Weight at study initiation: (P) Males: 257-304 g; Females: 175-213 g
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (Lot. No. 081110)
- Diet: Altromin 1324 maintenance diet for rats and mice (Lot. No. 1307), ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark/hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The amount of test substance for each dose concentration was suspended separately in aqua ad injectionem (sterile water) on each administration day, immediately before the administration. Homogeneity was ensured by using a vortex machine.

VEHICLE
- Justification for use and choice of vehicle: Selection was based on the solubility of the test item.
- Concentration in vehicle: 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no.: 0195A191

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- Females with unsuccessful mating were allowed to mate with other male of the same group.
- Females showing no evidence of copulation up to 14 day mating period were sacrificed 26 days after the last day of the mating period.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each concentration was analysed for nominal concentration. Homogeneity in the vehicle was analysed for the low and high dose concentrations. Samples for nominal concentration verification were taken in Week 1 (first week of pre-mating period), Week 3 (first week of mating), Week 5 (gestation) and Week 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the low and high dose preparation in Weeks 1 and 5.
All concentration samples were stored frozen (approx. -20 °C) until the analysis was performed.

Duration of treatment / exposure:

Males: 14 days before mating, 14 days during mating
Females: 14 days before mating, 14 days during mating, 20 days during gestation, 3 days during lactation

Frequency of treatment:

daily

Details on study schedule:

- Age at mating of the mated animals in the study: 12-13 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

other: yes, concurrent vehicle; the control group was shared with BSL Study no. 110998, another OECD 421 study, which was performed in parallel.

Details on study design:

- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. A descending sequence of dose levels was selected in order to demonstrate any dose-related response and a NOAEL.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily during weekend/holidays

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before assignment to the experimental groups and on the first day of administration.
Males were weighed weekly during the entire study period.
Females were weighed weekly during the pre-mating period, on Gestation Day 0, 7, 14, 20 and on Post-natal Day 0 (within 24 hours of parturition) and Post-natal Day 4 along with pups.

FOOD CONSUMPTION:
- Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration and was not measured during the mating period.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after the completion of mating period (Day 28)
- Maternal animals: All surviving animals were sacrificed on respective PND 4.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively:
ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), macroscopic lesions

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspringwere sacrificed at 4 days of age (PND 4).

The animals were subjected to postmortem examinations for gross external abnormalities.

Statistics:

One-way analysis of variance (ANOVA) followed by DUNETT’s multiple comparison test (p<0.05 was considered as statistical significant).

Reproductive indices:

Copulation Index (%) = (No. of rats copulated /No. of pairs) x 100
Fertility Index (%) = (No. of females pregant/No. of females copulated) x 100
Delivery Index(%) = (No. of dams with live newborns/ No. of pregnant dams) x 100

Offspring viability indices:

Viability Index (%) = (No. of live offspring at Day 4/ No. of live offspring at birth) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

histopathology correlates to discoloration in organs (see details below)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No treatment related clinical signs were observed. There were very few clinical signs recorded in control and treated animals during the study period but these findings were observed transiently post dose administration and had no relevance to treatment.
None of the animals died during the study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No effect on body weight was observed in both sexes throughout the complete study period.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS):
No treatment-related effects on testis or epididymidis weight were noted. No other sperm parameters were examined.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No treatment related effect on precoital interval and duration of gestation was observed.
The copulation index, fertility index and delivery index remained unaffected. No effects on mean No. of corporea lutea, No. of implantation sites, No. of live pups born, % pre- and post-implantation loss were observed.

ORGAN WEIGHTS (PARENTAL ANIMALS):
No statistically significant changes in absolute and relative organ weights were observed.

GROSS PATHOLOGY (PARENTAL ANIMALS):
In males, various macroscopic findings observed were yellow spots on epididymides (C:1/10, LD:1/10, MD:1/10), discoloration of epididymides (HD:5/10 animals), reddish discoloration of kidneys (LD: 4/10, MD: 6/10, HD: 10/10 animals), reddish discoloration of testes (HD: 3/10 animals), small testes and epididymides (HD: 1/10 animals).
In females, discoloration of kidneys (LD: 7/10, MD: 6/10, HD: 10/10 animals), cyst on kidneys (LD: 1/10 animal), ureter dilated (HD: 1/10 animal), dark discoloration of lung (HD: 1/10 animal), hardening of ovary (HD: 1/10 animal).
The discoloration of organs as well as reddish discoloration of the digestive tract noted in almost all treated animals is considered to be attributable to the colour of the test item and as such not a systemic effect due to the test item administration.

HISTOPATHOLOGY (PARENTAL ANIMALS):
There were foamy interstitial macrophages observed in testes of one male of MD group and in 9/10 males of HD group. There were no test item-related histological findings noted in other male and female reproductive organs.
Eosinophilic granules in the tubular epithelium of the renal cortex were seen in all animals of HD group and in a proportion of animals of MD group, but not in animals of LD group although macroscopically red discolored. This finding was considered to be due to test item deposition in renal tubuloepithelial cells. Moreover, in all females of the high dose group, renal corticotubular epithelium was diffusely vacuolated, possibly caused by washout of the test item during the histotechnical procedure.
The lung of one high dose female, being macroscopically dark discolored, showed minimal numbers of eosinophilic alveolar macrophages, considered to represent test item deposition.
All histological findings are due to test item deposition and there were no accompanying findings that would indicate organ damage.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects noted.

2

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects noted.

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING):
No treatment related effect was observed on total No. of pups born, No. of males, No. of females, sex ratio, live pups, still birth and runt on PND 0 and total No. of live pups and sex ratio on PND 4. Viability index was unaffected.

BODY WEIGHT (OFFSPRING):
No effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4.

GROSS PATHOLOGY (OFFSPRING):
No substance-related gross external findings were observed in any of the treated groups.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects noted.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Clinical Observations (P)

Clinical Findings	Control	100 mg/kg	300 mg/kg	1000 mg/kg
Males
Aggressive behavior	0/10	0/10	3/10	0/10
Nasal discharge	1/10	0/10	0/10	0/10
Piloerection	0/10	0/10	0/10	1/10
Salivation	0/10	0/10	1/10	0/10
Alopecia	1/10	0/10	1/10	0/10
Females
Aggressive behavior	1/10	0/10	0/10	0/10
Nasal discharge	0/10	2/10	0/10	0/10
Piloerection	0/10	1/10	0/10	1/10
Alopecia	0/10	1/10	2/10	2/10

 

Table 2: Macroscopic Findings (P)

Findings	Control	100 mg/kg	300 mg/kg	1000 mg/kg
Males
Epididymides: yellow spots	1/10	1/10	1/10	0/10
Epididymides: discoloration (reddish)	0/10	0/10	0/10	5/10
Epididymides: small	0/10	0/10	0/10	1/10
Kidney: discoloration (reddish)	0/10	4/10	6/10	10/10
Testes: discoloration (reddish)	0/10	0/10	0/10	3/10
Testes: small	0/10	0/10	0/10	1/10
Females
Kidney: discoloration (reddish)	0/10	7/10	6/10	10/10
Ureters: dilated	0/10	0/10	0/10	1/10
Lung: discoloration (dark)	0/10	0/10	0/10	1/10
Ovary: hard	0/10	0/10	0/10	1/10

 

Table 3: Reproductive Indices (%)

	Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Copulation Index	100	100	100	100
Fertility Index	60	80	70	60
Delivery Index	100	100	100	100
Viability Index	100	100	100	100

Applicant's summary and conclusion

Conclusions:

The test substance had no effect on reproductive performance.
Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40850/B TE, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg body weight for reproduction toxicity screening in males and females.
CLP: not classified
DSD: not classified

Executive summary:

Currently no study to assess the reproductive and/or developmental toxicity of Reactive Red 218 is available. Nevertheless, data from a Read across substance FAT 40850 is available. FAT 40850 was evaluated in a study performed according to OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

In this study, four groups comprised of 10 adult male and 10 non pregnant nulliparous female rats [Wistar Crl:WI(Han)] were dosed daily by oral gavage with 100, 300 and 1000 mg/kg body weight per day of FAT 40850/B TE at dose volume of 10 mL/kg body weight. The test item was formulated in sterile water with an administration volume of 10 mL/kg body weight. Control animals were handled identically as treated groups and received sterile water in similar volume as treated groups.

 

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period. After 14 days of treatment to both male and female, animals were paired (1:1) for maximum 14 days. The subsequent morning onwards, vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. Males and females were sacrificed on day 29 and post natal day 4 respectively and subjected to necropsy. Non pregnant females were sacrificed on their respective day 26 after the evidence of mating or completion of mating period.

 

There were no predominant clinical signs considered to be due to treatment observed in treatment groups compared to the controls. There were no mortalities observed in males or females during the study period. In males and females, statistical analysis of body weight data revealed no effect on body weight throughout the study period in treated groups when compared with controls.

 

There was no treatment related effect observed for duration of gestation and precoital interval in treated groups when compared with controls. All females in control and treated groups showed evidence of copulation during 14 days mating

period. Successful mating of females resulted in 60 % pregnancy rate each in the control and HD group, while 80% and 70% pregnancy rate in LD and MD groups, respectively.

The copulation index, fertility index and viability index in treated groups remained unaffected due to treatment when compared to the control. All pregnancies resulted in normal births and therefore delivery index remained unaffected in all treated groups. There was no treatment related effect observed on total number of pups born, number of male pups, number of female pups, sex ratio, live pups, still birth and runt on PND 0 and total number of live pups and sex ratio on PND 4 in treated groups compared to corresponding control. Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treated groups compared to the control.

 

In males and females, there were no treatment related changes observed in the absolute and relative organ weights of the treatment groups when compared with the controls. Histopathological evaluation showed there was no histomorphological indication of any functional impairment of the organs and tissues concerned.

 

Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40850/B TE, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg body weight for reproduction toxicity screening in males and females.