Reaction mass of 4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide and 4-[[4-(aminocarbonyl)phenyl]azo]-3-hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide

Endpoint summary

Description of key information

PR022

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed according to OECD TG 422. The test item was administered to Sprague Dawley rats (12/sex/dose) by gavage at dose levels of 0, 100, 300 and 1000 mg/kg/day for a total of 37 days (males) and through mating, gestation and delivery until day 4 of lactation (females). No toxic effects were observed in this study except increased liver weights in males and females of the highest dose group. As the effects on liver weight were minimal (less than 10% weight changes) and not accompanied by any histopathological or biochemical changes these effects were judged not to be adverse. The no-observed-adverse-effect level (NOAEL) for repeated dose toxicity was 1000 mg/kg under the conditions of this study. There were no effects on reproduction or development.

 

PR112

The toxicity of the test item when given by oral administration (gavage) to groups of 5 rats per sex per dose for 28 consecutive days at dosages of 0, 100, 300 or 1000 mg/kg bw/day, for 7 days/week have been investigated. No toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). Red staining of various body parts, red faeces and reddish contents of the gastro-intestinal tract noted among all groups treated with the test substance was considered to be related to staining properties of the test substance, and not to represent signs of systemic toxicity. No correlating histopathological abnormalities were noted. On the basis of these results, the high dose level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for the test substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (according to OECD 422 and GLP)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (Atsugi Breeding center)
- Age at study initiation: 8 weeks
- Weight at study initiation: males 309-355 g, females: 206 to 232 g
- Housing: individually; mating period: 1 male, 1 female; 1 litter during lactation
- Diet: autoclaved pellet diet for laboratory animals (CRF-1, Oriental Yeast Industry Co., LTD), ad libitum
- Water: sterile tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % sodium-carboxymethylcellulose (CMC-NA) aqueous solution containing 0.1 % Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of test substance : once or twice a week
- Storage temperature of suspension: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the first preparation, the dosing formulations were analyzed by HPLC and it was confirmed that the concentrations were within the target value +/-10%.

Duration of treatment / exposure:

males: from 14 days before mating and throughout the mating period until the day before necroscopy for a total of 37 days
females: from 14 days before mating, through the mating and gestation periods and delivery until day 4 of lactation for a total of 42 to 47 days. Non-delivering females, however, were treated until the day before necropsy.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: highest dose is the upper limit dose level stipulated in the OECD guideline
- Rationale for animal assignment (if not random): stratified-by-weight randomization method on the basis of the body weights to get almost the same mean body weights for each group.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before and after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: days 0,3,7 and 14 during the pre-mating period and once a week thereafter, days 0,7,14 and 20 of gestationand days 0 and 4 of lactation

FOOD CONSUMPTION: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to killing
- Anaesthetic used for blood collection: Yes (thiopental sodium)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte count, platelet count, prothrombin time, activated partial thromboplastin time, white blood cell count, differential leukocyte ratio and count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just priot to killing
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: ASAT, ALAT, gamma GT, ALP, total bilirubin, urea nitrogen, creatinine, glucose, total cholesterol, triglyeride, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium, potassium, chlorine


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On the day after the final dosing, all surviving males and females were euthanized by exsanguination from the abdominal aorta after blood sampling, and then were subjected to necropsy.

HISTOPATHOLOGY: Yes
The following organs/tissues of all animals were removed, and fixed and preserved in 10% neutral phosphate-buffered formalin. The testis and epididymis were fixed in Bouin's solution and preserved in 10% neutral phosphate-buffered formalin.
Brain, pituitary, thymus, lymphnode (mandibular and mesenteric), trachea, lung, stomach, intestines (duodenum, jejunum, ileum, cecum, colon, rectum), thyroid and parathyroid, heart, liver, spieen, kidney, adrenal, urinary bladder, testis, epididymis, seminal vesicle (including coagulating gland), ventral prostate, ovary, uterus, vagina, bone marrow (femur), sciatic nerve, spinal cord
As for the histopathological examination, the above-mentioned organs/tissues of all males and females in the control and 1000 mg/kg groups, ovary of non-pregnant females, and any gross lesions of all animals were stained with hematoxylin and eosin by standard methods, and were examined microscopically. However, both sides of the parathyroid could not be examined in 1 male in the control group, and only one side was examined in 4 males in the 1000 mg,/kg group because of loss of the specimen during the preparation.

Statistics:

Multiple comparison test:
Body weight, body weight gain, food consumption, hematology, blood chemistry, organ weight, number of corpora lutea, number of implantations, number of offspring

Kruskal-Wallis test and Dunnett-type multiple comparison test:
Days until copulation, number of estrus stages without copulation, mean estrous cycle length, gestation length, implantation index, delivery index, live birth index, incidence of offspring with external anomaly, viability index an Day 4

Chi-square test: Histopathological findings

Fisher's exact probability test:
Incidence of females with irregular estrous cycles, copulation index, fertility index, gestation index, sex ratio (rnale/fernale), incidence of dams with externally abnormal offspring

Clinical signs:

no effects observed

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 examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths were found in males or females in any group.
Reddish stool of a similar color to the test substance was observed every day from the day after the start of dosing in all males and females in the treated groups. Besides, transient loose stool was seen only an Day 22 of dosing in 2 males in the 1000 mg/kg group. However, there were no abnormal findings considered to be toxic changes in males or females in any group.

BODY WEIGHT AND WEIGHT GAIN
No significant differences were found in body weights or body weight gain of males or females between the control and treated groups throughout the observation period.

FOOD CONSUMPTION
No significant differences in food intake were found in males or females between the control and treated groups throughout the observation period.

HAEMATOLOGY
In males, a significant shortening of prothrombin time was observed in the 100 mg/kg group. However, it was judged to be incidental, because no change was detected in the 300 or 1000 mg/kg group.
In females, no significant differences were observed in any parameter between the control and treated groups.

CLINICAL CHEMISTRY
In males, A/G ratio in the 1000 mg/kg group was significantly higher than that in the control group. However, since it was a slight change without changes in total protein or albumin, this change was considered to be within physiological variations. Therefore, it was judged not treatment-related.
In females, ASAT (GOT) in the 100 mg/kg group was significantly higher than that in the control group. However, it was judged to be incidental, because no change was found in the 300 or 1000 mg/kg group.

ORGAN WEIGHTS
In males, significant increases in relative liver weights were noted in the 1000 mg/kg group. Besides, significantly lower absolute spleen weights were observed in the 300 mg/kg group, as were significantly higher relative adrenal weights in the 100 and 300 mg/kg groups. However, these changes were judged to be incidental, because no changes in their organs were found in the 1000 mg/kg group.
In females, significant increases in absolute and relative liver weights were noted in the 1000 mg/kg group. There were no changes in any Organs in the 100 or 300 mg/kg group.

GROSS PATHOLOGY
Changes considered to be attributable to the test substance were noted in the gastrointestinal tract.
Reddish-colored contents of a similar color to the test substance was observed in the stomach of 3 males in the 1000 mg/kg group, and the small and large intestines of all mal es and 2 females in the 100 mg/kg group and all males and females in the 300 and 1000 mg/kg groups. Besides, the following findings were observed; however they were judged to be incidental and not treatment-related, because there was no constant trend in their incidences: focal hemorrhage, incomplete contraction and white patch in the lung, tar-like contents in the stomach, white patch in the liver, dilatation of the renal pelvis, distention of the uterus and vagina, and dilatation of the cerebral ventricle.

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: no toxicological adverse effects observed up to highest dose tested

Critical effects observed:

not specified

There were no treatment related findings on reproduction. Details are presented in section 7.8.

Data on liver weight are presented in the following table:

 

Dose group (mg/kg bw/d)	Body weight (g)	Absolute liver weight (g)	Relative liver weight (g/100 g b.w.)	Animals per dose group
Females
0	309.9 +/- 20.9	10.486 +/_ 0.886	3.388 +/- 0.228	12
100	305.8 +/- 16.5	10.539 +/- 0.693	3.445 +/- 0.129	11
300	308.0 +/- 12.1	10.956 +/- 0.890	3.562 +/- 0.330	10
1000	306.1 +/- 25.5	11.414 +/- 0.775	3.743 +/- 0.271	12
Males
0	451.7 +/- 32.4	11.628 +/- 1.530	2.566 +/- 0.199	12
100	439.3 +/- 27.4	11.293 +/- 0.901	2.573 +/- 0.146	12
300	440.5 +/- 37.5	11.533 +/- 1.195	2.618 +/- 0.140	12
1000	458.0 +/- 33.5	12.650 +/- 1.429	2.759 +/- 0.179	12

data are presented as mean +/-SD

As the effects on liver weight were only minimal (less than 10%) and neither any changes in clinical chemistry indicative for liver damage nor any histopathological findings were observed in the liver, these effects on liver weight were regarded not to be adverse.

Conclusions:

Under the conditions of the test the only toxic effect observed was an increase in liver weights in males and females in the highest dose group (1000 mg/kg bw/d). As these effects were only minimal (less than 10% weight changes) and not accompanied by histopathological or biochemical changes they were judged not to be adverse. Therefore the no-observed-adverse-effect level (NOAEL) for repeated dose toxicity was 1000 mg/kg in both sexes, under the conditions of this study. There were no effects on reproduction or development.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed according to OECD TG 422. The test item was administered to Sprague Dawley rats (12/sex/dose) by gavage at dose levels of 0, 100, 300 and 1000 mg/kg/day for a total of 37 days (males) and through mating, gestation and delivery until day 4 of lactation (females). No toxic effects were observed in this study except increased liver weights in males and females of the highest dose group. As the effects on liver weight were minimal (less than 10% weight changes) and not accompanied by any histopathological or biochemical changes these effects were judged not to be adverse. The no-observed-adverse-effect level (NOAEL) for repeated dose toxicity was 1000 mg/kg under the conditions of this study. There were no effects on reproduction or development.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (according to OECD 422 and GLP)

Justification for type of information:

See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 210 (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (Atsugi Breeding center)
- Age at study initiation: 8 weeks
- Weight at study initiation: males 309-355 g, females: 206 to 232 g
- Housing: individually; mating period: 1 male, 1 female; 1 litter during lactation
- Diet: autoclaved pellet diet for laboratory animals (CRF-1, Oriental Yeast Industry Co., LTD), ad libitum
- Water: sterile tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % sodium-carboxymethylcellulose (CMC-NA) aqueous solution containing 0.1 % Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of test substance : once or twice a week
- Storage temperature of suspension: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the first preparation, the dosing formulations were analyzed by HPLC and it was confirmed that the concentrations were within the target value +/-10%.

Duration of treatment / exposure:

males: from 14 days before mating and throughout the mating period until the day before necroscopy for a total of 37 days
females: from 14 days before mating, through the mating and gestation periods and delivery until day 4 of lactation for a total of 42 to 47 days. Non-delivering females, however, were treated until the day before necropsy.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: highest dose is the upper limit dose level stipulated in the OECD guideline
- Rationale for animal assignment (if not random): stratified-by-weight randomization method on the basis of the body weights to get almost the same mean body weights for each group.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before and after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: days 0,3,7 and 14 during the pre-mating period and once a week thereafter, days 0,7,14 and 20 of gestationand days 0 and 4 of lactation

FOOD CONSUMPTION: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to killing
- Anaesthetic used for blood collection: Yes (thiopental sodium)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte count, platelet count, prothrombin time, activated partial thromboplastin time, white blood cell count, differential leukocyte ratio and count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just priot to killing
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: ASAT, ALAT, gamma GT, ALP, total bilirubin, urea nitrogen, creatinine, glucose, total cholesterol, triglyeride, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium, potassium, chlorine


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On the day after the final dosing, all surviving males and females were euthanized by exsanguination from the abdominal aorta after blood sampling, and then were subjected to necropsy.

HISTOPATHOLOGY: Yes
The following organs/tissues of all animals were removed, and fixed and preserved in 10% neutral phosphate-buffered formalin. The testis and epididymis were fixed in Bouin's solution and preserved in 10% neutral phosphate-buffered formalin.
Brain, pituitary, thymus, lymphnode (mandibular and mesenteric), trachea, lung, stomach, intestines (duodenum, jejunum, ileum, cecum, colon, rectum), thyroid and parathyroid, heart, liver, spieen, kidney, adrenal, urinary bladder, testis, epididymis, seminal vesicle (including coagulating gland), ventral prostate, ovary, uterus, vagina, bone marrow (femur), sciatic nerve, spinal cord
As for the histopathological examination, the above-mentioned organs/tissues of all males and females in the control and 1000 mg/kg groups, ovary of non-pregnant females, and any gross lesions of all animals were stained with hematoxylin and eosin by standard methods, and were examined microscopically. However, both sides of the parathyroid could not be examined in 1 male in the control group, and only one side was examined in 4 males in the 1000 mg,/kg group because of loss of the specimen during the preparation.

Statistics:

Multiple comparison test:
Body weight, body weight gain, food consumption, hematology, blood chemistry, organ weight, number of corpora lutea, number of implantations, number of offspring

Kruskal-Wallis test and Dunnett-type multiple comparison test:
Days until copulation, number of estrus stages without copulation, mean estrous cycle length, gestation length, implantation index, delivery index, live birth index, incidence of offspring with external anomaly, viability index an Day 4

Chi-square test: Histopathological findings

Fisher's exact probability test:
Incidence of females with irregular estrous cycles, copulation index, fertility index, gestation index, sex ratio (rnale/fernale), incidence of dams with externally abnormal offspring

Clinical signs:

no effects observed

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 examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths were found in males or females in any group.
Reddish stool of a similar color to the test substance was observed every day from the day after the start of dosing in all males and females in the treated groups. Besides, transient loose stool was seen only an Day 22 of dosing in 2 males in the 1000 mg/kg group. However, there were no abnormal findings considered to be toxic changes in males or females in any group.

BODY WEIGHT AND WEIGHT GAIN
No significant differences were found in body weights or body weight gain of males or females between the control and treated groups throughout the observation period.

FOOD CONSUMPTION
No significant differences in food intake were found in males or females between the control and treated groups throughout the observation period.

HAEMATOLOGY
In males, a significant shortening of prothrombin time was observed in the 100 mg/kg group. However, it was judged to be incidental, because no change was detected in the 300 or 1000 mg/kg group.
In females, no significant differences were observed in any parameter between the control and treated groups.

CLINICAL CHEMISTRY
In males, A/G ratio in the 1000 mg/kg group was significantly higher than that in the control group. However, since it was a slight change without changes in total protein or albumin, this change was considered to be within physiological variations. Therefore, it was judged not treatment-related.
In females, ASAT (GOT) in the 100 mg/kg group was significantly higher than that in the control group. However, it was judged to be incidental, because no change was found in the 300 or 1000 mg/kg group.

ORGAN WEIGHTS
In males, significant increases in relative liver weights were noted in the 1000 mg/kg group. Besides, significantly lower absolute spleen weights were observed in the 300 mg/kg group, as were significantly higher relative adrenal weights in the 100 and 300 mg/kg groups. However, these changes were judged to be incidental, because no changes in their organs were found in the 1000 mg/kg group.
In females, significant increases in absolute and relative liver weights were noted in the 1000 mg/kg group. There were no changes in any Organs in the 100 or 300 mg/kg group.

GROSS PATHOLOGY
Changes considered to be attributable to the test substance were noted in the gastrointestinal tract.
Reddish-colored contents of a similar color to the test substance was observed in the stomach of 3 males in the 1000 mg/kg group, and the small and large intestines of all mal es and 2 females in the 100 mg/kg group and all males and females in the 300 and 1000 mg/kg groups. Besides, the following findings were observed; however they were judged to be incidental and not treatment-related, because there was no constant trend in their incidences: focal hemorrhage, incomplete contraction and white patch in the lung, tar-like contents in the stomach, white patch in the liver, dilatation of the renal pelvis, distention of the uterus and vagina, and dilatation of the cerebral ventricle.

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: no toxicological adverse effects observed up to highest dose tested

Critical effects observed:

not specified

There were no treatment related findings on reproduction. Details are presented in section 7.8.

Data on liver weight are presented in the following table:

 

Dose group (mg/kg bw/d)	Body weight (g)	Absolute liver weight (g)	Relative liver weight (g/100 g b.w.)	Animals per dose group
Females
0	309.9 +/- 20.9	10.486 +/_ 0.886	3.388 +/- 0.228	12
100	305.8 +/- 16.5	10.539 +/- 0.693	3.445 +/- 0.129	11
300	308.0 +/- 12.1	10.956 +/- 0.890	3.562 +/- 0.330	10
1000	306.1 +/- 25.5	11.414 +/- 0.775	3.743 +/- 0.271	12
Males
0	451.7 +/- 32.4	11.628 +/- 1.530	2.566 +/- 0.199	12
100	439.3 +/- 27.4	11.293 +/- 0.901	2.573 +/- 0.146	12
300	440.5 +/- 37.5	11.533 +/- 1.195	2.618 +/- 0.140	12
1000	458.0 +/- 33.5	12.650 +/- 1.429	2.759 +/- 0.179	12

data are presented as mean +/-SD

As the effects on liver weight were only minimal (less than 10%) and neither any changes in clinical chemistry indicative for liver damage nor any histopathological findings were observed in the liver, these effects on liver weight were regarded not to be adverse.

Conclusions:

Under the conditions of the test the only toxic effect observed was an increase in liver weights in males and females in the highest dose group (1000 mg/kg bw/d). As these effects were only minimal (less than 10% weight changes) and not accompanied by histopathological or biochemical changes they were judged not to be adverse. Therefore the no-observed-adverse-effect level (NOAEL) for repeated dose toxicity was 1000 mg/kg in both sexes, under the conditions of this study. There were no effects on reproduction or development.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed according to OECD TG 422. The test item was administered to Sprague Dawley rats (12/sex/dose) by gavage at dose levels of 0, 100, 300 and 1000 mg/kg/day for a total of 37 days (males) and through mating, gestation and delivery until day 4 of lactation (females). No toxic effects were observed in this study except increased liver weights in males and females of the highest dose group. As the effects on liver weight were minimal (less than 10% weight changes) and not accompanied by any histopathological or biochemical changes these effects were judged not to be adverse. The no-observed-adverse-effect level (NOAEL) for repeated dose toxicity was 1000 mg/kg under the conditions of this study. There were no effects on reproduction or development.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 January 2008 to 30 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guidelines and according to GLP principles.

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males: 148-184 grams; females: 122-141 grams
- Fasting period before study: none
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in MIII type; height 15 cm.) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). No cage-enrichment was provided during overnight activity monitoring. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 °C (actual range: 19.9 – 21.5¿C),
- Humidity (%): 30-70% (actual range: 26 - 63%)
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 02 January 2008 to: 30 January 2008

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance formulations in propylene glycol formed a homogeneous suspension at the concentrations tested (coefficient of variation <10%). Analysis of the accuracy of dose preparations revealed values within the range of 102-106% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily for 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of a 5-day dose range finding study.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: mortality, viability
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily after dosing
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: subjective appraisal
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (iso-flurane anaesthesia)
- Animals fasted: Yes
- How many animals: 40
- Parameters examined: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets,
Clotting potential (Prothrombin time, Activated Partial thromboplastin time)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: 40
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine,
Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes:
At the end of week 1, 2, 3 and 4 of treatment, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength.
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerized monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were necropsied and descriptions of all macroscopic abnormalities recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution):
Identification marks: not processed
Ovaries, Adrenal glands, Pancreas, Aorta, Peyer's patches [jejunum, ileum] if detectable, Brain [cerebellum, mid-brain, cortex], Pituitary gland, Caecum,
(Preputial gland), Cervix, Prostate gland, (Clitoral gland), Rectum, Colon, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, (Eyes with optic nerve [if detectable] and (Skeletal muscle), Harderian gland), (Skin) , (Female mammary gland area), Spinal cord -cervical, midthoracic, lumbar, (Femur including joint), Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, (Larynx), Thyroid including parathyroid [if detectable], (Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, (Nasopharynx), Vagina, Oesophagus, All gross lesions

Tissues/organs mentioned in parentheses were not examined by the pathologist since there were no changes in macroscopic appearance indicative of (potential) toxicity.

The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOPATHOLOGY:
- all tissues collected at the scheduled sacrifice from all group 1 and 4 animals,
- all gross lesions.

Other examinations:

None

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test1 (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test2 (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test3 was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Clinical signs:

no effects observed

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:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Red staining of various body parts (shoulder, head, flank, back, neck, cervical region, chest
and/or generalized staining) and red faeces were noted among all groups treated with the test
substance.
Red staining of various body parts, red faeces and reddish contents of the gastro-intestinal tract
noted among all groups treated with the test substance was considered to be related to staining
properties of the test substance, and not to represent signs of systemic toxicity. No correlation
histopathological abnormalities were noted.

Incidental findings that were noted included rales, alopecia, scabs and a wound on the cheek.
These findings were considered to be within the normal range of clinical signs in rats of this age
and strain which are housed and treated under the conditions in this study. At the incidence
observed, these were considered signs of no toxicological significance.

No toxicologically significant changes in body weights and body weight gain were noted.
The statistically significant higher body weight gain of females at 300 and 1000 mg/kg/day in
week 2 occurred in the absence of a time- and dose-related response, and was of a very slight
nature. No toxicological relevance was ascribed to this change.

No toxicologically relevant changes occurred in haematological parameters of treated rats.
Any statistically significant changes in haematological parameters were considered to be of no
toxicological significance as they occurred in the absence of a treatment-related distribution,
remained within the range considered normal for rats of this age and strain and occurred in the
absence of supportive haematological changes. These changes consisted of higher reticulocyte
counts in males at 1000 mg/kg/day, higher methaemoglobin levels in females at 100 mg/kg/day
and higher, and lower mean corpuscular volume in females at 100 and 1000 mg/kg/day.

No toxicologically relevant changes occurredin clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of
no toxicological significance as they occurred in the absence of a treatment-related distribution
and remained within the range considered normal for rats of this age and strain. These changes
consisted of lower albumin levels in males at 300 mg/kg/day, higher cholesterol levels in males
at 100 mg/kg/day, higher total protein levels and lower urea levels in females at 100 mg/kg/day,
and lower inorganic phosphate levels in females at 100 and 1000 mg/kg/day.
A noticeably high aspartate aminotransferase activity level (ASAT) and glucose level was noted
for male no. 10 and 12 respectively. The incidence of these high levels was unrelated to the
dose, and therefore considered to be of no toxicological significance.

Reddish contents of the gastro-intestinal tract were observed among all animals treated with the
test substance.
Red staining of various body parts, red faeces and reddish contents of the gastro-intestinal tract
noted among all groups treated with the test substance was considered to be related to staining
properties of the test substance, and not to represent signs of systemic toxicity. No correlation
histopathological abnormalities were noted.

Incidental findings included a gray-white focus on the right lateral lobe of the liver, fluid in the
uterus and scab formation on the skin. These findings are occasionally seen among rats used in
these types of studies. In the absence of a treatment-related distribution they were considered
changes of no toxicological significance.

No toxicologically significant changes were noted in any of the parameters investigated in this
study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical
laboratory investigations, macroscopic examination, organ weights, and microscopic
examination).

From the results presented in this report a definitive No-Observed-(Adverse)-Effect Level
(NO(A)EL) for PIGMENT RED 112 of 1000 mg/kg/day was established.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No toxicologically significant changes were noted in any of the parameters investigated in this study.

Critical effects observed:

not specified

Conclusions:

The toxicity of the test item when given by oral administration (gavage) to groups of 5 rats per sex per dose for 28 consecutive days at dosages of 0, 100, 300 or 1000 mg/kg bw/day, for 7 days/week have been investigated. No toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). Red staining of various body parts, red faeces and reddish contents of the gastro-intestinal tract noted among all groups treated with the test substance was considered to be related to staining properties of the test substance, and not to represent signs of systemic toxicity. No correlating histopathological abnormalities were noted. On the basis of these results, the high dose level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for the test substance.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

02 January 2008 to 30 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guidelines and according to GLP principles.

Justification for type of information:

See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 210 (Chapter 13)

Reason / purpose for cross-reference:

read-across source

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males: 148-184 grams; females: 122-141 grams
- Fasting period before study: none
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in MIII type; height 15 cm.) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). No cage-enrichment was provided during overnight activity monitoring. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 °C (actual range: 19.9 – 21.5¿C),
- Humidity (%): 30-70% (actual range: 26 - 63%)
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 02 January 2008 to: 30 January 2008

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance formulations in propylene glycol formed a homogeneous suspension at the concentrations tested (coefficient of variation <10%). Analysis of the accuracy of dose preparations revealed values within the range of 102-106% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily for 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of a 5-day dose range finding study.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: mortality, viability
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily after dosing
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: subjective appraisal
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (iso-flurane anaesthesia)
- Animals fasted: Yes
- How many animals: 40
- Parameters examined: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets,
Clotting potential (Prothrombin time, Activated Partial thromboplastin time)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: 40
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine,
Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes:
At the end of week 1, 2, 3 and 4 of treatment, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength.
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerized monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were necropsied and descriptions of all macroscopic abnormalities recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution):
Identification marks: not processed
Ovaries, Adrenal glands, Pancreas, Aorta, Peyer's patches [jejunum, ileum] if detectable, Brain [cerebellum, mid-brain, cortex], Pituitary gland, Caecum,
(Preputial gland), Cervix, Prostate gland, (Clitoral gland), Rectum, Colon, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, (Eyes with optic nerve [if detectable] and (Skeletal muscle), Harderian gland), (Skin) , (Female mammary gland area), Spinal cord -cervical, midthoracic, lumbar, (Femur including joint), Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, (Larynx), Thyroid including parathyroid [if detectable], (Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, (Nasopharynx), Vagina, Oesophagus, All gross lesions

Tissues/organs mentioned in parentheses were not examined by the pathologist since there were no changes in macroscopic appearance indicative of (potential) toxicity.

The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOPATHOLOGY:
- all tissues collected at the scheduled sacrifice from all group 1 and 4 animals,
- all gross lesions.

Other examinations:

None

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test1 (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test2 (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test3 was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Clinical signs:

no effects observed

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:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Red staining of various body parts (shoulder, head, flank, back, neck, cervical region, chest
and/or generalized staining) and red faeces were noted among all groups treated with the test
substance.
Red staining of various body parts, red faeces and reddish contents of the gastro-intestinal tract
noted among all groups treated with the test substance was considered to be related to staining
properties of the test substance, and not to represent signs of systemic toxicity. No correlation
histopathological abnormalities were noted.

Incidental findings that were noted included rales, alopecia, scabs and a wound on the cheek.
These findings were considered to be within the normal range of clinical signs in rats of this age
and strain which are housed and treated under the conditions in this study. At the incidence
observed, these were considered signs of no toxicological significance.

No toxicologically significant changes in body weights and body weight gain were noted.
The statistically significant higher body weight gain of females at 300 and 1000 mg/kg/day in
week 2 occurred in the absence of a time- and dose-related response, and was of a very slight
nature. No toxicological relevance was ascribed to this change.

No toxicologically relevant changes occurred in haematological parameters of treated rats.
Any statistically significant changes in haematological parameters were considered to be of no
toxicological significance as they occurred in the absence of a treatment-related distribution,
remained within the range considered normal for rats of this age and strain and occurred in the
absence of supportive haematological changes. These changes consisted of higher reticulocyte
counts in males at 1000 mg/kg/day, higher methaemoglobin levels in females at 100 mg/kg/day
and higher, and lower mean corpuscular volume in females at 100 and 1000 mg/kg/day.

No toxicologically relevant changes occurredin clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of
no toxicological significance as they occurred in the absence of a treatment-related distribution
and remained within the range considered normal for rats of this age and strain. These changes
consisted of lower albumin levels in males at 300 mg/kg/day, higher cholesterol levels in males
at 100 mg/kg/day, higher total protein levels and lower urea levels in females at 100 mg/kg/day,
and lower inorganic phosphate levels in females at 100 and 1000 mg/kg/day.
A noticeably high aspartate aminotransferase activity level (ASAT) and glucose level was noted
for male no. 10 and 12 respectively. The incidence of these high levels was unrelated to the
dose, and therefore considered to be of no toxicological significance.

Reddish contents of the gastro-intestinal tract were observed among all animals treated with the
test substance.
Red staining of various body parts, red faeces and reddish contents of the gastro-intestinal tract
noted among all groups treated with the test substance was considered to be related to staining
properties of the test substance, and not to represent signs of systemic toxicity. No correlation
histopathological abnormalities were noted.

Incidental findings included a gray-white focus on the right lateral lobe of the liver, fluid in the
uterus and scab formation on the skin. These findings are occasionally seen among rats used in
these types of studies. In the absence of a treatment-related distribution they were considered
changes of no toxicological significance.

No toxicologically significant changes were noted in any of the parameters investigated in this
study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical
laboratory investigations, macroscopic examination, organ weights, and microscopic
examination).

From the results presented in this report a definitive No-Observed-(Adverse)-Effect Level
(NO(A)EL) for PIGMENT RED 112 of 1000 mg/kg/day was established.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No toxicologically significant changes were noted in any of the parameters investigated in this study.

Critical effects observed:

not specified

Conclusions:

The toxicity of the test item when given by oral administration (gavage) to groups of 5 rats per sex per dose for 28 consecutive days at dosages of 0, 100, 300 or 1000 mg/kg bw/day, for 7 days/week have been investigated. No toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). Red staining of various body parts, red faeces and reddish contents of the gastro-intestinal tract noted among all groups treated with the test substance was considered to be related to staining properties of the test substance, and not to represent signs of systemic toxicity. No correlating histopathological abnormalities were noted. On the basis of these results, the high dose level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification, as no relevant adverse effects were observed.