C.I. Acid Yellow 246

Endpoint summary

Description of key information

The 28-day repeated dose oral toxicity "no observed adverse effect level" (NOAEL) and "no-toxic-effect-level" (NOEL) of test substance is 1000 and 50 mg/kg bw respectively for male and females rats when administered by oral gavage while 28 day repeated dose dermal toxicity NOEL is 1000 mg/kg bw respectively for male and females rats.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 1987 to 06 April 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: FAT 20 306/B
Description: powder
Batch number: HT 2025/50
Purity: 96 %
Stability of test article: stable; expiration: year 2014
Stability of test article dilution: stable for at least 2 hours
Instructions for test article storage: room temperature

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Strain: Wistar rat, KFM-Han., outbred, SPF quality
- Source: Kleintierfarm Madoerin AG CH 4414 Fuellinsdorf/Switzerland
- Age at pretest: 7 - 8 weeks
- Weight at pretest: males: 153 - 174 g; females: 150 - 184 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard Kliba no. 343, Batch 73/87 and 77/87 rat maintenance diet ('Kliba', Klingentalmuehle AG, 4303 Kaiseraugst, Switzerland) ad libitum
- Water: Community tap water from Itingen was available ad libitum.
- Acclimation period: Seven days under laboratory conditions, after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light/12 hours dark, music/light period

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

4% (CMC)

Details on oral exposure:

TEST ARTICLE PREPARATION: The test article was weighed into a glass beaker on a tared Mettler PK 300 balance and the vehicle, carboxymethyl cellulose 4 % (CMC) in distilled water, was added. The mixture was prepared dally prior to administration using a homogenizer and kept stable during application with a magnetic stirrer.
Method: Oral by gavage, once daily. The control animals received the vehicle without the test article.
Frequency: Once daily, 7 days per week
Dose volume: 10 ml/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test article/vehicle mixture was determined during pretest. Intercurrent sampling for analyses was additionally performed during week 3 of test. Analyses were performed in the RCC Analytical Chemistry Laboratories, according to a method supplied by the sponsor.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days per week

Dose / conc.:

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

Remarks:

Control

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Middle dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

5/sex for test concentrations of 50 mg/kg and 200 mg/kg
10/sex for test concentrations of 0 mg/kg and 1000 mg/kg

Control animals:

yes, concurrent vehicle

Details on study design:

TEST ARTICLE PREPARATION
The test article was weighed into a glass beaker on a tared Mettler PK 300 balance and the vehicle, carboxymethyl cellulose 4 % (CMC) in distilled water, was added. The mixture was prepared dally prior to administration using a homogenizer and kept stable during application with a magnetic stirrer.

TEST METHOD
In this subacute toxicity study, test article was administered daily by gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. After termination of the treatment period animals were observed for a further 14 -day treatment-free recovery period.

Positive control:

no data

Observations and examinations performed and frequency:

VIABILITY / MORTALITY: Observations for mortality were recorded once daily.
CLINICAL SIGNS: Signs of toxicity were assessed once daily. Descriptions of all abnormalities were recorded, and the subsequent progress was monitored.
FOOD CONSUMPTION: The food consumption was recorded once during the acclimation period and weekly thereafter using an on-line electronic recording system consisting of a Mettler PK 4800 balance connected to the RCC computer.
BODY WEIGHTS: The body weight of each animal was recorded weekly during the acclimation and treatment period using an on-line electronic recording system consisting of a Mettler PK 4800 balance connected to the RCC computer.
OPHTHALMOSCOPIC EXAMINATIONS: at 4 weeks and at 6 weeks. Ophthalmoscopic examinations were performed on all animals. A description of any abnormality was recorded. Examinations were performed at termination of treatment and recovery period.

Sacrifice and pathology:

ORGAN WEIGHTS: The following organ weights were taken from all animals necropsied after 4 and 6 weeks respectively: Adrenals, Kidneys, Liver, Testes.
NECROPSY: after 4 weeks and after 6 weeks. All animals were necropsied, and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by a pathologist. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
HISTOTECHNOLOGY: All organ and tissue samples, as defined under Histopathology (following) were processed, embedded and cut at a thickness of 2 - 4 micrometers and stained with hematoxylin and eosin.
HISTOPATHOLOGY: Tissue samples of Adrenals, Heart, Kidneys, Liver, Spleen and Gross lesions collected at the end of the assigned study periods from the animals of the control and high-dose groups were examined by a pathologist.

Other examinations:

Clinical Laboratory Investigations:
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia
Blood and urine sampling: at 4 weeks and at 6 weeks

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) 1n the summary tables.

Clinical signs:

no effects observed

Description (incidence and severity):

Group 2 - 5 0 mg/kg:
One male animal (no. 12) showed slight sedation (days 17-18) and slight ruffled fur (days 17-29) and one female animal (no. 43) showed slight ruffled fur (days 17-29) during the treatment period. As shown above the animals had recovered from clinical signs at day 2 of the recovery period.
Group 3 - 200 mg/kg:
Four males (nos. 16, 18, 19 and 20) and two females (nos. 47 and 49) showed slight sedation, dyspnea, ruffled fur and alopecia at different days during weeks 2 to 4 and at day 29 (week 5, first day of recovery). The symptoms described were not all observed in the same animals as well as not all during the same observation period.
Group 4 - 1000 mg/kg:
Nine males (nos. 21-29) and ten females (nos. 51-60) showed slight respectively slight to moderate sedation, dyspnea, rales, ruffled fur, hunched posture, loss of weight and stiff gait. These symptoms were observed at different days during weeks 2 to 4. The intensity and duration varied among the animals. The animals which were kept for an additional recovery period of 14 days were not 1n all cases free of symptoms at termination of recovery. Nevertheless, most symptoms had recovered between days 2 to 10 of the treatment-free period.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related findings were noted at termination of treatment and/or recovery.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute kidney weights of the male rats of group 3 as well as kidney to body weight ratios of the male rats of group 4 were increased at termination of treatment period.
In addition, the liver weight ratios of the female rats of group 4 were reduced at termination of the recovery period.
These statistical differences were considered to be incidental and of no toxicological or biological relevance.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on study findings

Critical effects observed:

no

Conclusions:

The "no observed adverse effect level" (NOAEL) and "no-toxic-effect-level" (NOEL) of FAT 20306/B is 1000 and 50 mg/kg bw respectively for male and females rats when administered by oral gavage.

Executive summary:

In a GLP-compliant subacute 28-day toxicity study, FAT 20306/B was administered daily by gavage to Wistar rats according OECD guideline 407. The study was comprised of four groups (0, 50, 200 and 1000 mg/kg body weight) with a 14-day recovery period. Starting with day 17 of treatment one male and one female rat of the low-dose group showed slight sedation and ruffled fur. In addition, four males and two females of the mid-dose as well as nine males and ten females of the high-dose group 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. However, 2 days following exposure these symptoms disappeared (reversible).

No treatment-related effects were noted on all the parameters observed during the study such as mortality, food consumption, body weight, clinical signs, ophthalmoscopic examination, haematology, clinical chemistry, urine analysis, organ weight, gross and histo pathology.

Based upon the results the "no observed adverse effect level" (NOAEL) and  "no-toxic-effect-level"  (NOEL) of FAT 20306/B is 1000 and 50 mg/kg bw respectively for male and females rats when administered by oral gavage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant and guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 June 1985 to 16 October 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identification: FAT 20306/B
Description: powder
Batch No.: HT 2025/50
Purity: 97 % (guaranteed by the sponsor)
Stability of test article stable; expiration: 2014
Stability of test article in vehicle stable for at least 2 hours
Instruction for test article storage at room temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM
Species: Wistar Rat, KFM-Han., outbred, SPFquality
Rationale: Recognised by the international guidelines as the recommended test system.
Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland
Total number of animals: 20 males, 20 females
Age at start of treatment: males: 9 weeks, females: 11 weeks
Body weight range at start of treatment: males: 238 - 283 g, females: 208 - 252 g
Identification: By unique cage number and corresponding ear tag.
Randomization: Computer-generated random algorithm
Acclimation: One week under laboratory conditions, after veterinary examination
- Diet: standard Kliba 343, Batch 22/85 rat maintenance diet ("KLIBA"-Futter, Klingentalmuehle AG, Switzerland) ad libitum.
-Water: Community tap water from Itingen

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2
- Humidity (%): 55 ±10
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 hours of artificial fluorescent light each day. Music was played during light period.

Type of coverage:

occlusive

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

4 % dilution of CMC

Details on exposure:

Twenty-eight (28) dermal applications were administered to the shaved skin of the back. The skin area involved in the dermal application was equivalent to about 10 % of the total body surface. The hair was clipped from the dorsal surface once weekly throughout the study. The test article was applied evenly on the shaved skin and covered with an occlusive dressing. The dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage, for 6 hours per day, 7 days per week for a total of 28 application days. The application volume was 4 ml/kg body weight. The animals of the control group were treated with the vehicle alone in the same way (4 ml/kg body weight). Test article remainders were washed off with luke-warm tap water after termination of the daily treatment in animals of groups 2 to 4. They were dried with disposable paper towels.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

According to sponsor the stability and homogeneity of the test article/vehicle mixture has found to be stable if the mixture is prepared daily.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours per day, 7 days per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Middle dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Test Article Preparation: FAT 20306/B was weighed into a glass beaker on a tared Mettler PK 4800 balance and the vehicle added. The mixture was prepared using a homogenizer.
Frequency of Preparation: Daily prior to application.

Observations and examinations performed and frequency:

Symptoms (systemic): Twice daily
Symptoms (local): Daily prior to the following application according to Draize score.
Viability / Mortality: Twice daily
Food Consumption: Weekly
Body Weights: Twice weekly
Ophthalmoscopic Examinations: Observation by ophthalmoscope (Heine- Bifocal Ophthalmoscope, miroflex type) at termination of the application period

Sacrifice and pathology:

ORGAN WEIGHTS: The following organ weights were recorded on the scheduled date of necropsy: adrenal glands, ovaries, kidneys, testes, liver
NECROPSY AND HIST0PATH0L0GY: All animals mere necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by a pathologist. All animals were killed by intraperitoneal injection of sodium pentobarbital. Organs and tissues were fixed in 4 % neutral phosphate buffered formalin and embedded in Paraplast. Samples of the following tissues were collected from all animals a necropsy.

Other examinations:

CLINICAL Laboratory Investigations:
General:
Blood, samples for haematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for 18 hours before blood sampling, but water was provided. Blood samples were collected from each animal between the hours of 7.30 and 9.00 a.m. to reduce biologic variation caused by circadian rhythm. Blood samples were drawn from the retro-orbital plexus. Urine was collected into a specimen vial using a metabolism cage.
Blood and urine sampling: after 4 weeks. The assays of blood parameters were performed under quality control conditions to assure reliable test results.

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data :
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distri-bution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Description (incidence and severity):

Only one animal (female no. 39) of group 4, 1000 mg/kg showed slight erythema between day 4 and 8 and slight edema between day 4 and 8 of test on the treated skin.

Mortality:

no mortality observed

Description (incidence):

One female animal from group 04 died incidentally after blood sampling just before the regular autopsy. No treatment- related findings could be detected in this animal/ nor was an evident cause of death detectable.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

However, reduced body weight gain was noted in females of groups 3 and 4 during the last two weeks of treatment. These findings were related to the initial differences between the animals of the test article and control groups and not to test article treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

However, reduced food consumption values were noted in the female animals of groups 3 and 4. These findings were related to the initial differences betweeen the treated groups and the controls and not to test article treatment.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

no changes of toxicological significance after 4 weeks of treatment.

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The observed differences in female animals of groups 2, 3 and 4 were related to the significantly reduced final body weights, which were not related to test article treatment.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

mortality

ophthalmological examination

Remarks on result:

other: Only local findings, described as a slight edema and erythema, were observed in one animal of this qroup.

Critical effects observed:

not specified

Conclusions:

The "no-toxic effect level" (NOEL) of FAT 20306/B for 28 day dermal toxicity study in male and female rats is considered to be 1000 mg/kg bw/day.

Executive summary:

In a GLP-compliant dermal toxicity study with FAT 20306/B was conducted in Wistar rats for 6 hours/day (7 days/meek) for a total of 28 d applications according to OECD guideline 410 and EU method B.9. The study was comprised of 4 groups (0, 40, 200, 1000 mg/kg), for a total of 40 rats. No treatment-related effects were noted on most parameters observed during the study (mortality, food consumption, body weight, clinical signs, ophthalmoscopic examination, haematology, clinical chemistry, urine analysis, organ weight etc.). Only one animal (female no. 39) of group 4, 1000 mg/kg showed slight erythema between day 4 and 8 and slight edema between day 4 and 8 of test on the treated skin. No other local or systemic symptoms were observed in the animals of the test article-treated or control group. There were no adverse effects on gross and microscopic pathology. Based on the findings observed during this study the "no-toxic effect level" (NOEL) of FAT 20306/B for 28 day dermal toxicity study in male and female rats is considered to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-compliant guideline study

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 June 1985 to 16 October 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identification: FAT 20306/B
Description: powder
Batch No.: HT 2025/50
Purity: 97 % (guaranteed by the sponsor)
Stability of test article stable; expiration: 2014
Stability of test article in vehicle stable for at least 2 hours
Instruction for test article storage at room temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM
Species: Wistar Rat, KFM-Han., outbred, SPFquality
Rationale: Recognised by the international guidelines as the recommended test system.
Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland
Total number of animals: 20 males, 20 females
Age at start of treatment: males: 9 weeks, females: 11 weeks
Body weight range at start of treatment: males: 238 - 283 g, females: 208 - 252 g
Identification: By unique cage number and corresponding ear tag.
Randomization: Computer-generated random algorithm
Acclimation: One week under laboratory conditions, after veterinary examination
- Diet: standard Kliba 343, Batch 22/85 rat maintenance diet ("KLIBA"-Futter, Klingentalmuehle AG, Switzerland) ad libitum.
-Water: Community tap water from Itingen

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2
- Humidity (%): 55 ±10
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 hours of artificial fluorescent light each day. Music was played during light period.

Type of coverage:

occlusive

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

4 % dilution of CMC

Details on exposure:

Twenty-eight (28) dermal applications were administered to the shaved skin of the back. The skin area involved in the dermal application was equivalent to about 10 % of the total body surface. The hair was clipped from the dorsal surface once weekly throughout the study. The test article was applied evenly on the shaved skin and covered with an occlusive dressing. The dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage, for 6 hours per day, 7 days per week for a total of 28 application days. The application volume was 4 ml/kg body weight. The animals of the control group were treated with the vehicle alone in the same way (4 ml/kg body weight). Test article remainders were washed off with luke-warm tap water after termination of the daily treatment in animals of groups 2 to 4. They were dried with disposable paper towels.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

According to sponsor the stability and homogeneity of the test article/vehicle mixture has found to be stable if the mixture is prepared daily.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours per day, 7 days per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Middle dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Test Article Preparation: FAT 20306/B was weighed into a glass beaker on a tared Mettler PK 4800 balance and the vehicle added. The mixture was prepared using a homogenizer.
Frequency of Preparation: Daily prior to application.

Observations and examinations performed and frequency:

Symptoms (systemic): Twice daily
Symptoms (local): Daily prior to the following application according to Draize score.
Viability / Mortality: Twice daily
Food Consumption: Weekly
Body Weights: Twice weekly
Ophthalmoscopic Examinations: Observation by ophthalmoscope (Heine- Bifocal Ophthalmoscope, miroflex type) at termination of the application period

Sacrifice and pathology:

ORGAN WEIGHTS: The following organ weights were recorded on the scheduled date of necropsy: adrenal glands, ovaries, kidneys, testes, liver
NECROPSY AND HIST0PATH0L0GY: All animals mere necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by a pathologist. All animals were killed by intraperitoneal injection of sodium pentobarbital. Organs and tissues were fixed in 4 % neutral phosphate buffered formalin and embedded in Paraplast. Samples of the following tissues were collected from all animals a necropsy.

Other examinations:

CLINICAL Laboratory Investigations:
General:
Blood, samples for haematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for 18 hours before blood sampling, but water was provided. Blood samples were collected from each animal between the hours of 7.30 and 9.00 a.m. to reduce biologic variation caused by circadian rhythm. Blood samples were drawn from the retro-orbital plexus. Urine was collected into a specimen vial using a metabolism cage.
Blood and urine sampling: after 4 weeks. The assays of blood parameters were performed under quality control conditions to assure reliable test results.

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data :
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distri-bution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Description (incidence and severity):

Only one animal (female no. 39) of group 4, 1000 mg/kg showed slight erythema between day 4 and 8 and slight edema between day 4 and 8 of test on the treated skin.

Mortality:

no mortality observed

Description (incidence):

One female animal from group 04 died incidentally after blood sampling just before the regular autopsy. No treatment- related findings could be detected in this animal/ nor was an evident cause of death detectable.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

However, reduced body weight gain was noted in females of groups 3 and 4 during the last two weeks of treatment. These findings were related to the initial differences between the animals of the test article and control groups and not to test article treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

However, reduced food consumption values were noted in the female animals of groups 3 and 4. These findings were related to the initial differences betweeen the treated groups and the controls and not to test article treatment.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

no changes of toxicological significance after 4 weeks of treatment.

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The observed differences in female animals of groups 2, 3 and 4 were related to the significantly reduced final body weights, which were not related to test article treatment.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

mortality

ophthalmological examination

Remarks on result:

other: Only local findings, described as a slight edema and erythema, were observed in one animal of this qroup.

Critical effects observed:

not specified

Conclusions:

The "no-toxic effect level" (NOEL) of FAT 20306/B for 28 day dermal toxicity study in male and female rats is considered to be 1000 mg/kg bw/day.

Executive summary:

In a GLP-compliant dermal toxicity study with FAT 20306/B was conducted in Wistar rats for 6 hours/day (7 days/meek) for a total of 28 d applications according to OECD guideline 410 and EU method B.9. The study was comprised of 4 groups (0, 40, 200, 1000 mg/kg), for a total of 40 rats. No treatment-related effects were noted on most parameters observed during the study (mortality, food consumption, body weight, clinical signs, ophthalmoscopic examination, haematology, clinical chemistry, urine analysis, organ weight etc.). Only one animal (female no. 39) of group 4, 1000 mg/kg showed slight erythema between day 4 and 8 and slight edema between day 4 and 8 of test on the treated skin. No other local or systemic symptoms were observed in the animals of the test article-treated or control group. There were no adverse effects on gross and microscopic pathology. Based on the findings observed during this study the "no-toxic effect level" (NOEL) of FAT 20306/B for 28 day dermal toxicity study in male and female rats is considered to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Repeated dose toxicity: oral

In a GLP compliant subacute 28-day toxicity study, FAT 20306/B was administered daily by gavage to Wistar rats according OECD guideline 407. The study was comprised of four groups (0, 50, 200 and 1000 mg/kg body weight) with a 14-day recovery period. Starting with day 17 of treatment one male and one female rat of the low-dose group showed slight sedation and ruffled fur. In addition, four males and two females of the mid-dose as well as nine males and ten females of the high-dose group 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. However, 2 days following exposure these symptoms disappeared (reversible). No treatment-related effects were noted on all the parameters observed during the study such as mortality, food consumption, body weight, clinical signs, ophthalmoscopic examination, haematology, clinical chemistry, urine analysis, organ weight, gross and histo pathology. Based upon the results the "no observed adverse effect level" (NOAEL) and  "no-toxic-effect-level"  (NOEL) of FAT 20306/B is 1000 and 50 mg/kg bw respectively for male and females rats when administered by oral gavage.

 

Repeated dose toxicity: inhalation

Currently no study to assess the repeated dose inhalation toxicity potential of Acid Yellow 246 is available. However, the vapour pressure for the substance can be considered low owing to the high melting point (>287 °C). Hence, the substance is considered to have low volatility. Synthesis and spray drying of this chemical is performed in a closed process; the final product consists of non-dusty granules. Hence, the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Further, the chemical is found to have water solubility of >100 g/L and have low log partition coefficient (-0.77), hence in the case of dust of the substance entering the respiratory tract, it will be trapped in the mucus and cleared, thereby further limiting the absorption. The chemical showed low toxicity potential in the available acute oral and dermal toxicity studies (LD₅₀>2000 mg/kg bw) as well as no systemic toxicity in 5-days range finding study. No systemic toxicity was observed when Acid Yellow 246 was administered by gavage or applied dermally up to 1000 mg/kg bw/day in a 28-day repeated dose oral and dermal toxicity study. No elevated toxicity is expected from repeated dose inhalation exposure of Acid Yellow 246 and safety for human health can be estimated using the principles of route to route extrapolation. Hence, the conduct of repeated dose toxicity study via inhalation route for Acid Yellow 246 is considered to be scientifically not necessary.

 

Repeated dose toxicity: dermal

In a GLP compliant dermal toxicity study with FAT 20306/B was conducted in Wistar rats for 6 hours/day (7 days/meek) for a total of 28 d applications according to OECD guideline 410 and EU method B.9. The study was comprised of 4 groups (0, 40, 200, 1000 mg/kg), for a total of 40 rats. No treatment-related effects were noted on most parameters observed during the study (mortality, food consumption, body weight, clinical signs, ophthalmoscopic examination, haematology, clinical chemistry, urine analysis, organ weight etc.). Only one animal (female no. 39) of group 4, 1000 mg/kg showed slight erythema between day 4 and 8 and slight edema between day 4 and 8 of test on the treated skin. No other local or systemic symptoms were observed in the animals of the test article-treated or control group. There were no adverse effects on gross and microscopic pathology. Based on the findings observed during this study the "no-toxic effect level" (NOEL) of FAT 20306/B for 28 day dermal toxicity study in male and female rats is considered to be 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the findings of the repeated dose oral and dermal toxicity study, 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.