C.I. Acid Yellow 246

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The parental and reproduction No Observed Adverse Effect Level (NOAEL) of test substance was 400 mg/kg.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 2012 to 13 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 1995.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification FAT 20306/B
Molecular weight 517.59
CAS Number 119822-74-1
Description Red powder
Batch HT 2025/50
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 28 August 2015

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.

Rationale: This species and strain of rat has been recognized as appropriate for reproduction toxicity studies. WIL Research Europe B.V. has reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible
to the effects of reproductive toxicants.

Source F0: Charles River Deutschland, Sulzfeld, Germany.

Age at start F0-treatment: Approximately 11 weeks.

Number of F0-animals: 40 females and 40 males.

Acclimatization F0: At least 5 days prior to start of treatment.

Health inspection F0: Upon receipt of the animals.

Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20 % of the sex mean.

Identification F0: Earmark and tattoo.
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e., membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Number of pups: 445 pups.

Identification of pups On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
Animal husbandry
Room number: A0.13 (until Day 10 of study). A0.20 (from Day 10 of study onwards).

Conditions: Environmental controls for the animal room were set to maintain 18 to 24 °C, a relative humidity of 40 to 70 %, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation:
Pre-mating Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). Mating Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Lactation Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). General Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF®
Water: Free access to tap-water.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. Detection of mating was not confirmed for animal no. 70 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No test substance was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

The formulations of Group 2 and 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e., relative difference ≤10 %). The long-term storage samples were stable at ≤-70 °C for at least 9 days.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Details on study schedule:

Parental animals:
Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Frequency: Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Exposure period: Males were exposed for 28 days, i.e., 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-54 days, i.e., during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 43, 46 (Group 1), 55, 57 (Group 2), 72 and 79 (Group 4) were not dosed during littering.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Pups:
Pups were not dosed directly but could have potentially been exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Middle dose

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

HIgh dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose levels
Dose levels were based on a 28-day toxicity study in Wistar rats (RCC Project 092441) in which 50, 200 and 1000 mg/kg were tested. Starting with day 17 of treatment one male and one female rat at 50 mg/kg showed slight sedation and ruffled fur. In addition, four males and two females at 200 mg/kg as well as nine males and ten females at 1000 mg/kg showed dyspnea, ruffled fur, alopecia, sedation, rales, hunched posture, stiff gait and loss of weight. These symptoms were classified as slight to moderate. They were different in duration and intensity among the animals. A clear dose-relation was observed. During recovery period most of the described symptoms had recovered between days 2 to 10.

Parental animals: Observations and examinations:

Mortality / Viability: At least twice daily.

Clinical signs: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Oestrous cyclicity (parental animals):

Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.

Litter observations:

Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

Necropsy parental animals:
The animals were not deprived of food overnight.
Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Necropsy was conducted on the following days:

Condition:

Day of necropsy:
Females which delivered: Lactation Days 5-7.
Females which failed to deliver (nos. 63, 73 and 77)
Postcoitum Days 26-27 (females with evidence of mating)
Males: Following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Organ weights: The following organ weights and terminal body weight were recorded from all F0-males on the scheduled day of necropsy:
Epididymides:
Testes:

Histotechnology:
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2 - 4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of the males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5 - 7 of lactation. All pups were sexed, and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:

Observations/measurements in the study were recorded electronically using the following programs:
- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA).
- TOXDATA version 8.0 (WIL Research Europe B.V., ‘s-Hertogenbosch, The Netherlands): Mortality / Clinical signs / Body weights / Food consumption / Reproduction parameters / Observations pups / Organ weights.

Reproductive indices:

Mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care.

Offspring viability indices:

sex ratio

Clinical signs:

no effects observed

Description (incidence and severity):

Orange faeces noted for all animals treated at 400 mg/kg was considered due to the staining properties of the test substance, and not regarded toxicologically relevant.
Chromodacryorrhoea of the left eye was noted for one low dose male and alopecia was noted for one high dose female. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The statistically significantly increased mean body weight for females at 150 mg/kg on Day 14 postcoitum and decreased mean body weight gain for males at 50 mg/kg were not regarded toxicologically relevant as no dose response relationship was noted and the changes were very slight.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Sperm granuloma were recorded in the epididymides of two control group males, one male at 150 mg/kg and three males at 400 mg/kg and were the histologic correlates to the nodules noted at necropsy in this organ. There were no microscopic findings in any of the animals suspected of infertility which could explain their lack of reproductive performance.
The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for all males evaluated.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Reproduction data:
No toxicologically relevant effects on reproductive parameters were noted. There was one female at 150 mg/kg and two females at 400 mg/kg out of the ten mated animals per group which were not pregnant. These numbers were within normal limits. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Overall study finding

Clinical signs:

no effects observed

Description (incidence and severity):

Incidental clinical symptoms of pups consisted of blue snout, pale appearance, no milk in the stomach, and cold appearance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Two pups of the control group and five pups at 400 mg/kg were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. The statistically significantly effect on sex ratio at 50 mg/kg was not considered toxicologically relevant as the sex ratio was considered normal and no dose response relationship was noted.


Observations:
There was one pup (litter 79; Group 4) with missing tail on Day 4 of lactation, which was confirmed at macroscopic examination during planned necropsy on Day 5 of lactation.
The nature and incidence of these findings remained within the range considered normal for pups of this age and were therefore considered to be of no toxicological relevance.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Overall study finding

Critical effects observed:

not specified

Reproductive effects observed:

no

Conclusions:

The parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of FAT 20306/B was 400 mg/kg.

Executive summary:

A GLP-compliant reproduction/developmental toxicity screening test of FAT 20306/B in Wistar rats by oral gavage was carried out according to e study OECD guideline 421. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 50, 150 and 400 mg/kg. Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. The observations and examinations evaluated were mortality / viability, clinical signs (daily), body weight and food consumption (at least at weekly intervals), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability. Orange faeces noted for all animals treated at 400 mg/kg was considered due to the staining properties of the test substance, and not regarded toxicologically relevant. The statistically significantly increased mean body weight for females at 150 mg/kg on Day 14 postcoitum and decreased mean body weight gain for males at 50 mg/kg were not regarded toxicologically relevant as no dose response relationship was noted and the changes were very slight. No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. There were no parental, reproduction and developmental toxicity was observed up to 400 mg/kg so based on the study findings the parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 400 mg/kg was derived.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Only this study is available, and the study is Klimisch code 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A GLP-compliant reproduction/developmental toxicity screening test of FAT 20306/B in Wistar rats by oral gavage was carried out according to e study OECD guideline 421. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 50, 150 and 400 mg/kg. Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Orange faeces noted for all animals treated at 400 mg/kg was considered due to the staining properties of the test substance, and not regarded toxicologically relevant. The statistically significantly increased mean body weight for females at 150 mg/kg on Day 14 postcoitum and decreased mean body weight gain for males at 50 mg/kg were not regarded toxicologically relevant as no dose response relationship was noted and the changes were very slight. No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. There were no parental, reproduction and developmental toxicity was observed up to 400 mg/kg so based on the study findings the parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 400 mg/kg was derived.

Effects on developmental toxicity

Description of key information

The developmental No Observed Adverse Effect Level (NOAEL) of test substance was 400 mg/kg.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

OECD 421

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 2012 to 13 March 2013

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:

other: REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification FAT 20306/B
Molecular weight 517.59
CAS Number 119822-74-1
Description Red powder
Batch HT 2025/50
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 28 August 2015

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.

Rationale: This species and strain of rat has been recognized as appropriate for reproduction toxicity studies. WIL Research Europe B.V. has reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible
to the effects of reproductive toxicants.

Source F0: Charles River Deutschland, Sulzfeld, Germany.

Age at start F0-treatment Approximately 11 weeks.

Number of F0-animals: 40 females and 40 males.

Acclimatization F0: At least 5 days prior to start of treatment.

Health inspection F0: Upon receipt of the animals.

Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20 % of the sex mean.

Identification F0: Earmark and tattoo.
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e., membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Number of pups: 445 pups.

Identification of pups On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
Animal husbandry
Room number: A0.13 (until Day 10 of study). A0.20 (from Day 10 of study onwards).

Conditions. Environmental controls for the animal room were set to maintain 18 to 24 °C, a relative humidity of 40 to 70 %, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation:
Pre-mating Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Lactation Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). General Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF®
Water: Free access to tap-water.

Route of administration:

oral: gavage

Details on exposure:

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No test substance was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with target concentrations (i.e., mean accuracies between 90 % and 110 %). The formulations of Group 2 and 4 were homogeneous (i.e., coefficient of variation ≤10 %). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e., relative difference ≤10 %). The long-term storage samples were stable at ≤-70 °C for at least 9 days.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. Detection of mating was not confirmed for animal no. 70 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e., 2 weeks prior to mating, during mating, and up to termination.
Females were exposed for 41 - 54 days, i.e., during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Duration of test:

Males were exposed for 28 days, i.e., 2 weeks prior to mating, during mating, and up to termination.
Females were exposed for 41 - 54 days, i.e., during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Middle dose

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose levels:
Dose levels were based on a 28-day toxicity study in Wistar rats (RCC Project 092441) in which 50, 200 and 1000 mg/kg were tested. Starting with day 17 of treatment one male and one female rat at 50 mg/kg showed slight sedation and ruffled fur. In addition, four males and two females at 200 mg/kg as well as nine males and ten females at 1000 mg/kg showed dyspnea, ruffled fur, alopecia, sedation, rales, hunched posture, stiff gait and loss of weight. These symptoms were classified as slight to moderate. They were different in duration and intensity among the animals. A clear dose-relation was observed. During recovery period most of the described symptoms had recovered between days 2 to 10.

Maternal examinations:

Mortality / Viability At least twice daily.

Clinical signs Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body weights Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Fetal examinations:

Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Statistics:

Observations/measurements in the study were recorded electronically using the following programs:
- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA).
- TOXDATA version 8.0 (WIL Research Europe B.V., ‘s-Hertogenbosch, The Netherlands): Mortality / Clinical signs / Body weights / Food consumption / Reproduction parameters / Observations pups / Organ weights.

Indices:

Mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio.

Clinical signs:

no effects observed

Description (incidence and severity):

Orange faeces noted for all animals treated at 400 mg/kg was considered due to the staining properties of the test substance, and not regarded toxicologically relevant.
Chromodacryorrhoea of the left eye was noted for one low dose male and alopecia was noted for one high dose female. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The statistically significantly increased mean body weight for females at 150 mg/kg on Day 14 postcoitum and decreased mean body weight gain for males at 50 mg/kg were not regarded toxicologically relevant as no dose response relationship was noted and the changes were very slight.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

There was one animal with nodule(s) in the epididymides in the control group, one at 150 mg/kg and one at 400 mg/kg.
The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included foci on the thymus, pelvic dilation of the kidneys, alopecia, and discolouration of the clitoral glands.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Sperm granuloma were recorded in the epididymides of two control group males, one male at 150 mg/kg and three males at 400 mg/kg, and were the histologic correlates to the nodules noted at necropsy in this organ.
There were no microscopic findings in any of the animals suspected of infertility which could explain their lack of reproductive performance.
The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for all males evaluated.

Pre- and post-implantation loss:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Reproduction data:
No toxicologically relevant effects on reproductive parameters were noted.

There was one female at 150 mg/kg and two females at 400 mg/kg out of the ten mated animals per group which were not pregnant. These numbers were within normal limits.
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Overall study finding

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Incidental clinical symptoms of pups consisted of blue snout, pale appearance, no milk in the stomach, and cold appearance. Two pups of the control group and five pups at 400 mg/kg were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age. No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. The statistically significantly effect on sex ratio at 50 mg/kg was not considered toxicologically relevant as the sex ratio was considered normal and no dose response relationship was noted.

Observations:
There was one pup (litter 79; Group 4) with missing tail on Day 4 of lactation, which was confirmed at macroscopic examination during planned necropsy on Day 5 of lactation.
The nature and incidence of these findings remained within the range considered normal for pups of this age and were therefore considered to be of no toxicological relevance.

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Overall study finding

Abnormalities:

not specified

Developmental effects observed:

no

Conclusions:

The parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of FAT 20306/B was 400 mg/kg.

Executive summary:

A GLP-compliant reproduction/developmental toxicity screening test of FAT 20206/B in rats by oral gavage was carried out according to OECD guideline 421, in which four groups of ten male and ten female Wistar rats received the test substance at 0, 50, 150 and 400 mg/kg bw/day via oral gavage. Apart from investigations into reproductive parameters, the study design also included investigations into developmental parameters including early postnatal pup development (mortality, clinical signs, body weights and macroscopy). No toxicologically relevant effects on reproductive parameters were noted in this study. The administration of the test substance to maternal animals did not adversely affect the gestation index and duration, parturition and maternal care. No treatment-related effects on pup development such as mortality, clinical signs and body weight of pups were seen. Also, no macroscopic findings indicative of developmental toxicity were reported. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 400 mg/kg bw/day was derived.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 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

In the reproduction/developmental toxicity screening test discussed under 'Reproductive toxicity section', the study design also included investigations into developmental parameters including early postnatal pup development (mortality, clinical signs, body weights and macroscopy). No toxicologically relevant effects on reproductive parameters were noted in this study. The administration of the test substance to maternal animals did not adversely affect the gestation index and duration, parturition and maternal care. No treatment related effects on mortality, clinical signs and body weight of pups were seen. Also, no macroscopic findings indicative of developmental toxicity were reported. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 400 mg/kg was derived.

Justification for classification or non-classification

Based on the results of a reproductive toxicity screening study, no effects were seen on parental toxicity, fertility or development and thus the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.