C.I. Reactive Yellow 161

Endpoint summary

Description of key information

Oral: no study available.

Inhalation: no study available.

Dermal: The test item was used in an 28-d repeated dose toxicity study with male and female rats according to OECD 410. The no adverse effect level NOAEL in male rats was determined at 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

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:

1984

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Rent, England
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 53 ± 5 days
- Weight at study initiation: 160 to 197 g
- Fasting period before study: NA
- Housing: individually
- Diet (ad libitum): Scientific Feeds LAD 1 diet obtained from Special Diets Services Limited, 1, Stepfield, Witham, Essex, England
- Water (ad libitum): tap water
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY: analytically verified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Animal room temperature was controlled at a mean minimum of 22.3°C and a mean maximum of 24.9°C
- Humidity (%): Relative humidity was not controlled but was recorded daily using a.wet and dry bulb thermometer (mean 48.0 % RH)
- Air changes (per hr): Air exchange was maintained at a rate of approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was controlled to give 12 hours artificial light in each 24 hour period

IN-LIFE DATES: The study commenced on 11 April 1984 and the rats were killed on 9 May 1984.

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

Treatment procedure
The hair was clipped from the mid-dorsal region of each rat exposing an area of skin equivalent to approximately 10% of the total body surface. Hair clipping was carried out approximately twenty-four hours prior to the first application of the test substance.
Hair clipping was repeated as necessary during the experimental period.
The skin sites were not abraded.

The test substance, at the concentration supplied by the Sponsor, was spread evenly over the treatment area of each rat of Groups 2 to 4 inclusive at the appropriate dosage level and moistened with distilled water.
The treatment site was covered with an impervious bandage consisting of gauze covered with 'Elastoplast' elastic adhesive dressing backed with impervious 'Sleek' plaster.
The test substance remained on the back of each rat for a total of 6 hours each day, after which time the dressing was removed and the treated skin washed with warm (30-40°C) water and gently blotted dry.
Control animals were similarly dosed with distilled water.
Each animal received a constant dosage level based upon its most recent bodyweight.
Animals were treated once daily, seven days per week for four weeks.

Duration of treatment / exposure:

6 hours/day for 4 weeks

Frequency of treatment:

daily

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Dosage levels of 40, 200 and 1000 mg/kg/day of FAT 40 162B were selected on the basis of acute oral and dermal toxicity data in rats (LD50 > 5000 mg/kg bodyweight and > 2000 mg/kg bodyweight respectively) which was supplied by the Sponsor and a preliminary 5-day dermal Irritation study in rabbits conducted at Huntingdon Research Centre (Report No. CBG 364P/845761).

Observations and examinations performed and frequency:

Clinical signs
All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.
All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. This allowed a post mortem examination to be undertaken during the working part of that day. At weekends and public holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Any animal showing signs of severe debility or toxicosis was isolated, and if found to be in extremis was killed.

Local dermal irritation
Local irritation was recorded immediately prior to the first daily application of the test substance and subsequently daily. Local dermal reactions (erythema and oedema) resulting from treatment were assessed on a numerical basis according to a modified Draize scoring system, as follows:
Erythema and eschar Formation:
No erythema 0
Slight erythema 1
Well defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedema formation:
No oedema 0
Slight oedema 1
Well defined oedema (area well defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4

Bodyweights
All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

Food consumption
The quantity of food consumed by each rat was measured at weekly intervals throughout the study.

Clinical laboratory studies
Removal of blood samples
Food was withdrawn overnight prior to collection of samples. Blood was withdrawn under light ether anaesthesia from the orbital sinus of all rats prior to termination (week 4).

Haematology
Estimations were performed by Technicon Hemalog 8/90 Micro, using standard Technicon methodology.
Packed cell volume (PCV)
Haemoglobin (Hb)
Red blood cell count (RBC)
Platelet count (plts)
Total white cell count (WBC)
Differential count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes)
Mean corpuscular haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Thrombocytes

Clinical chemistry
The following estimations were performed by Roche Cobas Bio CentrifUgal analyser:
Glucose
Alkaline phosphatase (AP) -
Glutamic-pyruvic transaminase (GPT), also known as alanine aminotransferase.
Glutamic-oxaloacetic transaminase (GOT), also known as aspartate aminotransferase
Total bilirubin

The following estimations were performed by Technicon SMA 12/60 using standard Technicon methodology:
Urea (transformed to blood urea nitrogen by calculation
Total protein
Albumin
Globulin (by subtraction) A/G ratio (by caiculation)
Sodium (Na+)
Potassium (X+)
Chloride (Cl)7
Calcium (Ca++)
Inorganic Phosphorus (P--)
Creatinine
Cholesterol (Technicon enzymatic methodology)

Sacrifice and pathology:

All animals dying during the study or killed in extremis were autopsied and, when possible, tissues were examined histologically. After 28 days of treatment (day 29) all remaining animals were randomly killed by carbon dioxide asphyxiation and a complete autopsy undertaken. The macroscopic appearance of the tissues was recorded.
The following organs from each animal killed after four weeks were dissected free of fat and weighed:
adrenals
ovaries
kidneys
testes (with epididymides)
liver

Samples of the following tissues from all rats in the control and high dosage groups were preserved in 10% buffered formalin for subsequent histological examination:
kidneys
liver
skin (treated and untreated)
any other macroscopically abnormal tissue

Statistics:

Statistical analyses were applied to the following parameters; bodyweight, food consumption, haematology, clinical chemistry and organ weights.
Analysis of variance followed by Student's 't' test, Williams' test or, where necessary, Kruskal-Wallis analysis, were used to assess the significance of intergroup differences. Where appropriate, values were log transformed to stabilise variance.
Analysis of organ weights was perforrned using analysis of covariance. Organ weights were adjusted for final bodyweight as covariate where this was the more efficient method of analysis. Where appropriate, organ weights were log transformed to stabilise variance. Group means were compared using Student's 't' test or a non-parametric equivalent.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female rat no. 28 of group 2 (40 mg/kg/day) died after blood sampling on Day 23 and female rat no. 36 of group 4 (1000 mg/kg/day) died during anaesthesia on the same day. No abnormalities considered to be related to treatment were observed prior to death or at post mortem examination in either animal. A smell of ether in the thoracic cavity was noted in both rats. The cause of death was considered to have been anaesthetic overdose.

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically ignificantly decreased Na+ and cholesterol levels (P<0.05 using Williams' test) were recorded in male rats of groups 3 and 4 when compared with the controls at week 4. A marginal increase (P(0.05) in Cl- levels was also noted in male rats of group 4 (1000 mg/kg/day); this resulted from the high individual value recorded for rat 16d (1000 mg/kg/day) and the low value recorded in rat 5d (Control - distilled water).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decreased kidney weights (P<0.01 using Williams' test) were recorded in male rats of group 4 (1000 mg/kg/day) when compared with controls. No morphological changes were apparent in the kidneys of male rats of the high dosage group at histological examination.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

No relevant adverse effects which can be contributed to the test substance were observed during the entire study period.

Executive summary:

Reactive Yellow 161 was administered to the skin of rats, daily ät dosage levels of 40, 200 and 1000 mg/kg/day for 28 consecutive days.

Bodyweight gains of male and female rats receiving the test substance at all dosage levels were comparable with those of the controls.

The food consumption of female rats of all three treatment groups was significantly less than that of the controls during week 1 only.

At week 4, significantly decreased Na+ and cholesterol levels were recorded in male rats treated with Reactive Yellow 161 at 200 and 1000 mg/kg/day when compared with the controls.

Significantly decreased kidney weights were recorded in male rats of group 4 (1000 mg/kg/day) in comparison with control values.

No morphological changes were apparent at histological examination of the kidneys.

In all other respects including general health bodyweight gain, haematology, macroscopic and microscopic pathology, rats receiving Reactive Yellow 161 were comparable to the controls.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Organ:

kidney

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:

1984

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Rent, England
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 53 ± 5 days
- Weight at study initiation: 160 to 197 g
- Fasting period before study: NA
- Housing: individually
- Diet (ad libitum): Scientific Feeds LAD 1 diet obtained from Special Diets Services Limited, 1, Stepfield, Witham, Essex, England
- Water (ad libitum): tap water
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY: analytically verified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Animal room temperature was controlled at a mean minimum of 22.3°C and a mean maximum of 24.9°C
- Humidity (%): Relative humidity was not controlled but was recorded daily using a.wet and dry bulb thermometer (mean 48.0 % RH)
- Air changes (per hr): Air exchange was maintained at a rate of approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was controlled to give 12 hours artificial light in each 24 hour period

IN-LIFE DATES: The study commenced on 11 April 1984 and the rats were killed on 9 May 1984.

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

Treatment procedure
The hair was clipped from the mid-dorsal region of each rat exposing an area of skin equivalent to approximately 10% of the total body surface. Hair clipping was carried out approximately twenty-four hours prior to the first application of the test substance.
Hair clipping was repeated as necessary during the experimental period.
The skin sites were not abraded.

The test substance, at the concentration supplied by the Sponsor, was spread evenly over the treatment area of each rat of Groups 2 to 4 inclusive at the appropriate dosage level and moistened with distilled water.
The treatment site was covered with an impervious bandage consisting of gauze covered with 'Elastoplast' elastic adhesive dressing backed with impervious 'Sleek' plaster.
The test substance remained on the back of each rat for a total of 6 hours each day, after which time the dressing was removed and the treated skin washed with warm (30-40°C) water and gently blotted dry.
Control animals were similarly dosed with distilled water.
Each animal received a constant dosage level based upon its most recent bodyweight.
Animals were treated once daily, seven days per week for four weeks.

Duration of treatment / exposure:

6 hours/day for 4 weeks

Frequency of treatment:

daily

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Dosage levels of 40, 200 and 1000 mg/kg/day of FAT 40 162B were selected on the basis of acute oral and dermal toxicity data in rats (LD50 > 5000 mg/kg bodyweight and > 2000 mg/kg bodyweight respectively) which was supplied by the Sponsor and a preliminary 5-day dermal Irritation study in rabbits conducted at Huntingdon Research Centre (Report No. CBG 364P/845761).

Observations and examinations performed and frequency:

Clinical signs
All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.
All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. This allowed a post mortem examination to be undertaken during the working part of that day. At weekends and public holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Any animal showing signs of severe debility or toxicosis was isolated, and if found to be in extremis was killed.

Local dermal irritation
Local irritation was recorded immediately prior to the first daily application of the test substance and subsequently daily. Local dermal reactions (erythema and oedema) resulting from treatment were assessed on a numerical basis according to a modified Draize scoring system, as follows:
Erythema and eschar Formation:
No erythema 0
Slight erythema 1
Well defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedema formation:
No oedema 0
Slight oedema 1
Well defined oedema (area well defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4

Bodyweights
All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

Food consumption
The quantity of food consumed by each rat was measured at weekly intervals throughout the study.

Clinical laboratory studies
Removal of blood samples
Food was withdrawn overnight prior to collection of samples. Blood was withdrawn under light ether anaesthesia from the orbital sinus of all rats prior to termination (week 4).

Haematology
Estimations were performed by Technicon Hemalog 8/90 Micro, using standard Technicon methodology.
Packed cell volume (PCV)
Haemoglobin (Hb)
Red blood cell count (RBC)
Platelet count (plts)
Total white cell count (WBC)
Differential count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes)
Mean corpuscular haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Thrombocytes

Clinical chemistry
The following estimations were performed by Roche Cobas Bio CentrifUgal analyser:
Glucose
Alkaline phosphatase (AP) -
Glutamic-pyruvic transaminase (GPT), also known as alanine aminotransferase.
Glutamic-oxaloacetic transaminase (GOT), also known as aspartate aminotransferase
Total bilirubin

The following estimations were performed by Technicon SMA 12/60 using standard Technicon methodology:
Urea (transformed to blood urea nitrogen by calculation
Total protein
Albumin
Globulin (by subtraction) A/G ratio (by caiculation)
Sodium (Na+)
Potassium (X+)
Chloride (Cl)7
Calcium (Ca++)
Inorganic Phosphorus (P--)
Creatinine
Cholesterol (Technicon enzymatic methodology)

Sacrifice and pathology:

All animals dying during the study or killed in extremis were autopsied and, when possible, tissues were examined histologically. After 28 days of treatment (day 29) all remaining animals were randomly killed by carbon dioxide asphyxiation and a complete autopsy undertaken. The macroscopic appearance of the tissues was recorded.
The following organs from each animal killed after four weeks were dissected free of fat and weighed:
adrenals
ovaries
kidneys
testes (with epididymides)
liver

Samples of the following tissues from all rats in the control and high dosage groups were preserved in 10% buffered formalin for subsequent histological examination:
kidneys
liver
skin (treated and untreated)
any other macroscopically abnormal tissue

Statistics:

Statistical analyses were applied to the following parameters; bodyweight, food consumption, haematology, clinical chemistry and organ weights.
Analysis of variance followed by Student's 't' test, Williams' test or, where necessary, Kruskal-Wallis analysis, were used to assess the significance of intergroup differences. Where appropriate, values were log transformed to stabilise variance.
Analysis of organ weights was perforrned using analysis of covariance. Organ weights were adjusted for final bodyweight as covariate where this was the more efficient method of analysis. Where appropriate, organ weights were log transformed to stabilise variance. Group means were compared using Student's 't' test or a non-parametric equivalent.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female rat no. 28 of group 2 (40 mg/kg/day) died after blood sampling on Day 23 and female rat no. 36 of group 4 (1000 mg/kg/day) died during anaesthesia on the same day. No abnormalities considered to be related to treatment were observed prior to death or at post mortem examination in either animal. A smell of ether in the thoracic cavity was noted in both rats. The cause of death was considered to have been anaesthetic overdose.

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically ignificantly decreased Na+ and cholesterol levels (P<0.05 using Williams' test) were recorded in male rats of groups 3 and 4 when compared with the controls at week 4. A marginal increase (P(0.05) in Cl- levels was also noted in male rats of group 4 (1000 mg/kg/day); this resulted from the high individual value recorded for rat 16d (1000 mg/kg/day) and the low value recorded in rat 5d (Control - distilled water).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decreased kidney weights (P<0.01 using Williams' test) were recorded in male rats of group 4 (1000 mg/kg/day) when compared with controls. No morphological changes were apparent in the kidneys of male rats of the high dosage group at histological examination.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

No relevant adverse effects which can be contributed to the test substance were observed during the entire study period.

Executive summary:

Reactive Yellow 161 was administered to the skin of rats, daily ät dosage levels of 40, 200 and 1000 mg/kg/day for 28 consecutive days.

Bodyweight gains of male and female rats receiving the test substance at all dosage levels were comparable with those of the controls.

The food consumption of female rats of all three treatment groups was significantly less than that of the controls during week 1 only.

At week 4, significantly decreased Na+ and cholesterol levels were recorded in male rats treated with Reactive Yellow 161 at 200 and 1000 mg/kg/day when compared with the controls.

Significantly decreased kidney weights were recorded in male rats of group 4 (1000 mg/kg/day) in comparison with control values.

No morphological changes were apparent at histological examination of the kidneys.

In all other respects including general health bodyweight gain, haematology, macroscopic and microscopic pathology, rats receiving Reactive Yellow 161 were comparable to the controls.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat