Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOAEL for reproductive toxicity was determined to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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:
guideline study
Justification for type of information:
Refer chapter 13 for the detailed analogue justification.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 1995
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 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.
CLP: not classified
DSD: not classified
Executive summary:

In a study performed according to OECD Guideline 421, four groups comprising of 10 adult male and 10 non pregnant nulliparous female rats [Wistar Crl:WI(Han)] were dosed daily by oral gavage with 0, 100, 300 and 1000 mg/kg bw/day of REACTIVE RED 286 at dose volume of 10 mL/kg bw.  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 high dose group, while 80 % and 70 % pregnancy rate in low and mid dose groups, respectively. The copulation, fertility and viability indices 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 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. Hence, based on the above findings, the no observed adverse effect level (NOAEL) for REACTIVE RED 286 is believed to be 1000 mg/kg bw/day for reproductive as well as developmental toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality study conducted according to OECD TG 421 and in compliance to GLP
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Currently no study to assess the reproductive and/or developmental toxicity of FAT 40034 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.

Effects on developmental toxicity

Description of key information

The NOAEL for developmental toxicity was determined to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Nov 2010 - 23 Dec 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Refer chapter 13 for detailed analogue justification.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: Females: 167-201 g, (mean: 184.78 g, ± 20% = ± 36.96 g); Males: 286-318 g, (mean: 297.98 g, ± 20% = ± 59.60 g)
- Housing: The animals were housed individually in IVC cages (except during mating period when 2 females were paired with one male), type III H, polysulphone cages on Altromin saw fiber bedding (Lot No.: 050810, 040810, 150910, 021010 and 070111)
- Diet (ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice (Lot No.: 1315 and 1013)
- Water (ad libitum): Free access to tap water, acidified using sulfuric acid to a pH of approximately 2.8
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

IN-LIFE DATES: From: 29 Nov 2010 To: 23 Dec 2010
Route of administration:
oral: gavage
Vehicle:
other: Sterile water (Manufacturer: Delta Select)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in sterile water. The vehicle was chosen based on the test item’s solubility. The test item formulation was prepared freshly on each administration day before the administration procedure.
The test substance as well as the vehicle was administered at a dose volume of 10 mL/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed on samples collected at at various intervals.
Samples for analysis of the dose formulations of the test item in the vehicle (nominal concentration) were taken in the first and last week of the study for all doses.
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first and last week of the study.
All formulation samples were stored at -20 °C and analyzed after completion of the in-life phase of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 103763.
Details on mating procedure:
After acclimatisation, females were paired with males as per the ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to regularize the number of animals for terminal sacrifice on particular day. The subsequent morning and the next morning onwards, the vaginal smear of female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as Gestation Day 0. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other.
Duration of treatment / exposure:
Gestation Day 5-19
Frequency of treatment:
Daily
Duration of test:
Animals were sacrificed on Gestation Day 20.
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: According to the results of the dose range finding study (BSL Study No. 103757).
Maternal examinations:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed twice daily during the entire gestation period except during weekends and holidays where clinical observation was made only once. Mortality, morbidity, pertinent behavioural changes and all signs of overt toxicity were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the day of receipt to ensure that the body weights were within the ± 20% variation.
The sperm positive females were weighed during GD 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except on the day of receipt.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption of pregnant females was measured on GD 5, 8, 11, 14, 17 and 20. The food consumption was presented for period 0-5, 5-8, 8-11, 11-14, 14-17, and 17-20. The food consumption was measured neither for males during the entire study nor for both male and females during the mating period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 20
- Examinations: At the time of termination, the presumed pregnant females were examined macroscopically for any structural abnormalities or pathological changes which might have influenced the pregnancy.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: All fetuses from particular dams were identified by different colored strings, weighed and sexed, based on the anogenital distance. Each fetus was examined for external anomalies.
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one interval to the next.
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control and test groups. Fetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Chi-square test.The statistical analysis was performed with GraphPad Prism (Version V) software (p<0.05 was considered as statistical significant).
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical Observation:
No test item related clinical signs were observed in any of the dose group females during the entire period of the treatment when compared with controls. The clinical signs observed in control group were wet nose and mouth (2/25), alopecia (1/25); in LD group was alopecia (1/25); in MD group were wet nose and mouth (1/25), alopecia (2/25), piloerection (2/25), nasal discharge (1/25); in HD group were alopecia (3/25), piloerection (7/25), nasal discharge (1/25), salivation (7/25), moving the bedding (10/25), respiratory sound (2/25). These symptoms were observed transiently occurring immediately after dose administration and are not assumed to be related to systemic toxicity. All animals survived throughout the study period.

Body Weight and Body Weight Change:
No test item related effect on body weight and body weight change was observed during the treatment period. However, statistical analysis of body weight and body weight change data revealed significant decrease for body weight in HD group on GD 5 and body weight change in LD group between GD 8-11. The statistically significant decrease observed in HD and LD groups were considered incidental and did not have any toxicological relevance.

Food Consumption:
Statistical analysis revealed no treatment related effect on food consumption during treatment period in any of the treatment groups when compared with corresponding controls.

Prenatal Data:
Statistical analysis of prenatal data revealed no significant effect on parameters.

Litter Data:
Statistical analysis of litter data revealed no significant effect on parameters.
The pregnancy rate observed in different groups is as follows, Control group (96%), LD (84%), MD (100%) and HD (88%).

Gross Pathology:
The terminally sacrificed animals belonging to the control and LD group revealed no findings, but there were few lesions observed in MD or HD groups like spleen enlarged (MD group: 1/25; HD group 0/25), lung with red spots ( MD group: 1/25 and HD groups: 1/25) and discolored kidney (MD group: 0/25; HD group 3/25). These finding were observed in few individual animals and were not considered treatment related.
The pattern of gross pathological lesions at necropsy in very few animals from different treatment groups suggested that the lesions were of spontaneous/incidental in nature.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Few external abnormalities were seen among the control and treatment groups. Predominant external finding noted was haematoma on various body locations in control and treated groups. These finding in treatment group were statistically insignificant compared to controls.

Skeletal examination of the Alizarin red stained fetuses revealed a range of abnormalities which were of a type or which occurred at an incidence comparable in treated groups when compared with controls. Statistically significant increase in incidence was observed for incomplete ossification of 4th metacarpal-bilateral (HD group), Xiphoid (MD group) and parietal (LD group) and significant decrease in incidences was observed for dumbbell shaped-4th sternebrum (HD group) and rudimentary 14th rib–bilateral (HD group). The incidence incomplete ossification of 4th metacarpal-bilateral was observed in one isolated animal of HD group and was considered incidental. However, the statistical significant difference observed for the findings revealed lack of dose dependency and indicated no relevance with treatment. There was no indication of a compound related trend in the type and incidences of other abnormalities and they were therefore considered to be spontaneous in nature.

Internal observation of the fetal viscera by free hand microdissection technique revealed a range of visceral abnormalities in all groups including control. There was statistically significant increase in heart hyperemia (without the aneurysmal changes) in MD and HD groups compared to controls. This finding is reflected as the consequence of a functional disorder and do not represent the developmental anomalies. However, due to very less severity and lack of dose dependency this incidence was classified as variation. The other visceral abnormalities observed in treated groups were in frequencies comparable or even less in numbers to controls, therefore no toxicological significance can be attributed to these findings and considered to be spontaneous in nature.

Craniofacial examination by razor blade serial sectioning technique revealed a range of visceral abnormalities in all groups including controls. These findings were in frequencies comparable or even less in numbers to controls. Therefore, these findings are not to be considered as treatment related and solely spontaneous in nature.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No treatment related adverse effects were observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
The NOAEL for both maternal toxicity and fetal toxicity of FAT 40850/B TE in Wistar rats was 1000 mg/kg bw/day.
Executive summary:

This prenatal developmental toxicity study of FAT 40850/B TE was conducted in pregnant female Wistar rats to detect possible adverse effects on pregnant females and embryofetal development when administered by oral gavage from Gestation Day 5 to 19. Nulliparous and non pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smear (GD 0). Four groups of presumed pregnant females 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. Control animals were handled identically as treated groups and received aqua ad injectionem (sterile water) as a vehicle in similar volume as treated groups.

The test item formulation was prepared freshly and dose volumes were adjusted based on the most recent body weight measurement.

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured on various gestation days.

The treated and control females were sacrificed on respective Gestation Day 20. Followed by the gross necropsy evaluation of the females, the uteri and ovaries were removed, weighed and examined for number of corpora lutea, implantations, resorptions (early and late), live and dead fetuses. Fetuses were identified by color strings, sexed and weighed. All fetuses were observed for external abnormalities. Half of the foetuses were observed for the visceral and craniofacial abnormalities and remaing half of the litter for skeletal abnormalities.

Uteri of the non pregnant females were processed with 10% ammonium sulphide solution and checked for the early embryonic deaths if any.

There were few clinical signs observed in females of control and treatment groups during the period of the treatment. The clinical signs recorded during the study in the treatment groups were observed transiently and were not considered to be due to systemic effect of the test item.

Body weight, body weight change and food consumption remained unaffected throughout the treatment period.

Statistical analysis of prenatal data and litter data revealed no significant effect on parameters like terminal body weight, gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantations, live fetuses, group mean number of male and female fetuses, total fetuses, sex ratio, fetal positions, dead fetuses, group litter mean weight, male and female litter weight, resorptions, percent preimplantation loss and post implantation loss in the treated groups when compared with controls.

The pregnancy rate observed in different groups is as follows, Control group (96%), LD (84%), MD (100%) and HD (88%).

Few gross external abnormalities were seen among the control and treatment groups. Haematoma on various body locations were the predominant findings observed in both control and treatment groups.

Skeletal examination of the Alizarin red stained fetuses revealed a range of abnormalities, which were of a type or which occurred at an incidence comparable in treated groups when compared with controls. Statistically significant increase in incidence was observed for incomplete ossification of 4th metacarpal-bilateral (HD group), Xiphoid (MD group) and parietal (LD group) and significant decrease in incidences was observed for dumbbell shaped-4th sternebrum (HD group) and rudimentary 14th rib –bilateral (HD group).

Internal observation of the fetal viscera by free hand microdissection technique revealed a range of visceral abnormalities in all groups including control. There was statistically significant increase in heart hyperemia (without the aneurysmal changes) in MD and HD groups. This finding was observed with very less severity and showed lack of dose dependency.

Craniofacial examination by razor blade serial sectioning technique revealed a range of visceral abnormalities in all groups including controls.

The terminally sacrificed animals belonging to the control and LD group revealed no findings, but there were lesions like spleen enlarged, lung with red spots and discolored kidney observed in MD or HD groups. These finding were observed in few individual animals and were not considered treatment related.

In conclusion, the repeated dose administration of FAT 40850/B TE to pregnant female Wistar rats at dosages of 100, 300 and 1000 mg/kg bw/day from Gestation Day 5 to 19 revealed no major toxicological findings in females and fetuses.

Based on the data generated from this study, the NOAEL for both maternal toxicity and fetal toxicity of FAT 40850/B TE in Wistar rats was 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality study conducted according to OECD TG 421 and in compliance to GLP
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 a prenatal developmental toxicity study conducted according to OECD Guideline 414, pregnant female Wistar rats were administered REACTIVE RED 286 at the doses of 0, 100, 300 and 1000 mg/kg bw/day from gestation Day 5 to 19. Statistical analysis of prenatal data and litter data revealed no significant effect on parameters like terminal body weight, gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantations, live fetuses, group mean number of male and female fetuses, total fetuses, sex ratio, fetal positions, dead fetuses, group litter mean weight, male and female litter weight, resorptions, percent preimplantation loss and post implantation loss in the treated groups when compared with controls. The pregnancy rate in different groups, control (96 %), low (84 %), mid (100 %) and high dose (88 %) also remained unaffected. Skeletal examination of the Alizarin red stained fetuses revealed a range of abnormalities which were of a type or which occurred at an incidence comparable in treated groups when compared with controls. Statistically significant increase in incidence was observed for incomplete ossification of 4th metacarpal-bilateral (high dose), Xiphoid (mid dose) and parietal (low dose) and significant decrease in incidences was observed for dumbbell shaped-4th sternebrum (high dose) and rudimentary 14th rib–bilateral (high dose). The incidence incomplete ossification of 4th metacarpal-bilateral was observed in one isolated animal of high dose group and was considered incidental. However, the statistical significant difference observed for the findings revealed lack of dose dependency and indicated no relevance with treatment. There was no indication of a compound related trend in the type and incidences of other abnormalities and they were therefore considered to be spontaneous in nature. Internal observation of the fetal viscera by free hand microdissection technique revealed a range of visceral abnormalities in all groups including control. There was statistically significant increase in heart hyperemia (without the aneurysmal changes) in mid and high dose groups compared to controls. This finding is reflected as the consequence of a functional disorder and do not represent the developmental anomalies. However, due to very less severity and lack of dose dependency this incidence was classified as variation. The other visceral abnormalities observed in treated groups were in frequencies comparable or even less in numbers to controls, therefore no toxicological significance can be attributed to these findings and considered to be spontaneous in nature. Craniofacial examination by razor blade serial sectioning technique revealed a range of visceral abnormalities in all groups including controls. These findings were in frequencies comparable or even less in numbers to controls. Therefore, these findings were not considered as treatment related but solely spontaneous in nature. In conclusion, the repeated administration of REACTIVE RED 286 to pregnant female Wistar rats at dosages of 100, 300 and 1000 mg/kg bw/day from Gestation Day 5 to 19 revealed no major toxicological findings in females and foetuses, hence the NOAEL for both maternal as well as developmental toxicity for REACTIVE RED 286 in Wistar rats was 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data, FAT 40034 does not warrant the classification as per the CLP (EU regulation No. 1272/2008) criteria.