Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Endpoint summary

Description of key information

Repeated dose toxicity: oral

Data on short-term repeat dose oral toxicity study was not available for the target substance Reactive Red 024:1. To fill the data gaps, read-across approach is adapted using similar substanceReactive Red 286(FAT 40850).Read-across is claimed basis of structural relationship of the target and the source chemicals. Read-across substance have been investigated for 28-day repeated dose oral toxicity.Oral administration (gavage) of FAT 40850/A to Wistar rats at doses of 100, 300 and 1000 mg/kg/day, for 28 days resulted in no deaths and no test item related changes in daily or weekly observations or the functional observational battery (performed during week 4) including grip strength and locomotor activity. The study was performed under GLP conditions and in accordance with EU Method B.7. No findings were noted concerning the estrous stage of the female animals.

The following test item related findings were noted:

- Red stained feces, noted in males and females at all dose levels (100, 300 and 1000 mg/kg/day) were considered to be caused by a dyeing effect of the test item but not to be adverse. Red discoloration was also detected macroscopically in multiple organs, like kidneys, mesenteric lymphnodes and stomach.

- A significant decrease in the white blood cell count and in lymphocytes (absolute) with values below ranges of historical control data in males at 1000 mg/kg/day was possibly related to the treatment with the test item. The lymphocyte value in males treated with 300 mg/kg/day was also decreased and there seemed to be a tendency in reduction of lymphocytes (absolute) with an effect on total WBC in the highest dose group. In the absence of histopathologigal changes e.g. in the spleen, bone marrow, thymus or lymphnodes and no findings in the females, this effect is considered not to be adverse.

- A decrease in potassium and phosphorus concentrations was noted in males at 1000 mg/kg/day and, as occurred in the high dose group, is considered to be test item-related, although potassium levels are within the range of historical data. Furthermore, the total protein level was increased in correlation with increased albumin and increased albumin/globulin ratio. As these findings were noted only in males and as there was no dose response relationship, these changes were considered not to be of adverse nature.

- The increase in liver weights is considered to be caused by an adaptive reaction but, in absence of further findings, e.g. pathohistological changes in the liver, not to be of adverse nature.

- Changes in kidney weights in males at 1000 mg/kg/day are in correlation with macroscopic and microscopic findings. Histologically, treatment related findings consisted of minimal to slight deposits of a red pigment in the kidneys of animals of both sexes treated with 1000 mg/kg/day and in females treated with 300 mg/kg/day. The pigment was red fine granular and located in the tubular epithelium, and sometimes associated with increased severity of hyaline droplets in males treated with 1000 mg/kg/day and minimal to slight vacuolar degeneration of tubular epithelium in females of the same dose group. The deposits of red pigment were considered to be not adverse itself, but the secondary reactions consisting of increased severity of hyaline droplets and vacuolar degeneration of tubular epithelium were deemed to be adverse. Based on the read across data generated from the 28-day repeated dose oral toxicity with FAT 40850/A in Wistar rats, the no observed adverse effect level (NOAEL) of target substance Reactive Red 024:1 is also considered to be 300 mg/kg/d.

Repeated dose toxicity: inhalation

However, the vapour pressure for the substance can be considered low owing to the high melting point (>350 °C), hence, the substance is considered to have low volatility. Synthesis and formulation of this chemical is performed in a closed process; the final product consists of liquid formulations only. 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. Based on column 2, ‘Specific rules for adaptation from column 1’ of the table given in REACH Annex VII, the study on repeat dose inhalation toxicity only needs to be conducted if an exposure via inhalation is to be expected, based on vapour pressure and/or the likelihood of an exposure to aerosols, particles or droplets. Referring to the expected low volatility of the substance, the fact that the substance is imported into the EU in a formulated form as a dust-free powder or as a granulate, the exposure via inhalation is unlikely occur. Further, Reactive Red 024:1 was found to be miscible in water (water solubility 434 g/L) and have low log partition coefficient <-3.52), 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 inhalation toxicity study (LC₅₀>1500 mg/m³) and also in acute oral toxicity study (LD₅₀>5000 mg/kg), with no mortality or systemic toxicity.Further, no adverse effects were observed in a 28-day oral repeated dose toxicity study up to 300 mg/kg bw (with the source substance). Hence, safety for human health can be estimated via route to route extrapolation. Taking above arguments into account, low toxicity potential is expected on repeated exposure of Reactive Red 024:1 via inhalation route and hence testing by the inhalation route was considered scientifically not necessary.

Repeated dose toxicity: dermal

Currently no study to assess repeated dose dermal toxicity of FAT 40034 is available. However, the molecular weight of the substance is 802.1 g/mol, which indicates substance is too large for dermal absorption. Further, high water solubility (434 g/L) and low partition coefficient (-3.52), indicate the substance may be too hydrophilic to cross the lipid rich environment of thestratum corneum. Hence, the dermal uptake for the substance is expected to be low. Further results of exposure to test animals via the oral route in a repeated dose toxicity study with source substance are available, hence no elevated toxicity other than seen in this study, is expected via the dermal route and safety for human health can be estimated via route to route extrapolation. Similarly absence of systemic toxicity or mortality in skin irritation as well as sensitization studies with FAT 40034, further supports the conclusion that no additional adverse effects other than seen in repeated dose oral toxicity study with source substance are expected via dermal route. Further experience with similar chemical substances has demonstrated that it is very unlikely that toxicity related to the intrinsic properties of the substance only show up upon dermal exposure and not after systemic application. Taking into consideration all the above arguments, further experiments to assess repeated dose dermal toxicity are not taken into account.

Key value for chemical safety assessment

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:

2010-08-17 till 2010-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: TZ 5978 / BOP 07-09
- Expiration date of the lot/batch: 31-Jul-2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the original container at room temperature (15 - 25 °C)
- Stability of the test substance in the solvent/dispersant/vehicle/test medium: Up to 8 days at room temperature (15 - 25 °C)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rationale: Recognized by international guidelines as a recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Rat, RccHanTM: WIST(SPF)
- Source:Harlan Laboratories B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
- Age at study initiation:Ca. 7 weeks
- Weight at study initiation: Males: 182 to 207 g, Females: 148 to 168 g
- Fasting period before study: no data
- Housing:In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding including paper enrichment
- Diet: Pelleted standard Harlan Teklad 2914C rat / mouse maintenance diet was available ad libitum
- Water:Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 5 days. Under test conditions after health examination. Only animals without any visible signs of illness were used for the study
- Pre-Randomization Period: 1 day
- Randomization: Randomly allocated to groups by body weight
- Group 10: reserve, not used in the test


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Values outside of these ranges occasionally occurred, usually following room cleaning, and are considered not to have any influence on the study.
- Air changes (per hr):Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12-hour fluorescent light/12-hour dark cycle with music during the light period.

Route of administration:

oral: gavage

Details on route of administration:

Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.

Vehicle:

water

Remarks:

bidistilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DOSING SOLUTIONS PREPARATION
- Rate of preparation of dosing preparation (frequency):The dose formulations were prepared weekly.
FAT 40850/A TE was weighed into a glass beaker on a tared Mettler balance and a part of the vehicle (approx. one third) was added, stirred and the
remaining vehicle was added (except the first preparation, when in the low dose the total volume of the vehicle was added). The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 - 25 °C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
- Stability of dose formulations: stable in application formulations when kept four hours and eight days under room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.
- Storage of dose formulations: in glass beakers at room temperature (15 - 25 °C).


VEHICLE
- Justification for use and choice of vehicle (if other than water): none (water)
- Concentration in vehicle: 0 mg/mL, 10 mg/mL, 30 mg/mL and 100 mg/mL
- Amount of vehicle (if gavage): Dose Volume: 10 mL/kg body weight
- Purity: bidistilled water
- Dose Volume: 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis was performed by Harlan Laboratories Ltd. using a HPLC method provided by the Sponsor. After experimental start and during week 3, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 h and 8 days). The samples were delivered to the analytical laboratory (Harlan Laboratories Ltd., Analytics, Itingen / Switzerland) at room temperature (20 ± 5 °C) and stored there at -20 ± 5 °C until analysis. The application formulations investigated during the study were found to comprise FAT 40850/A in the range of 93.3 % to 111.3 % and, thus, the required content limit of ±20 % with reference to the nominal concentration was met. The homogeneous distribution of FAT 40850/A in the preparations was approved because single results found did not deviate more than 5.6 % (<15 %) from the corresponding mean. The following acceptance criteria will be applied to analytical results:
sample contents should be within a range of ±20 % of nominal content. Formulations will be considered homogeneous if the maximum deviation from mean calculated from top, middle and bottom samples is not more than 15 %. The results obtained from storage stability samples should not deviate more than 10 % fromtime-zero reference (content or mean of homogeneity samples).

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily, 7d/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

300 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:

- Dose selection rationale:The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats, Harlan Laboratories study C93571 (non-GLP).
- Rationale for animal assignment (if not random):Randomly allocated to groups by body weight.
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):Sacrifice: After 4 Weeks. All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. Slides of all organs and tissues collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. Additionally, the kidneys of both sexes were examined from the animals of the low and middle dose groups to establish a no-effect level.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table No1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:The animals were observed for clinical signs once daily during acclimatization as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period.

Weekly Behavioral Observations
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations:Body weights were recorded once during the pre-randomization phase, once weekly during the acclimatization and treatment periods and once before necropsy,

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, the food consumption was recorded once during the acclimatization period and weekly thereafter
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


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, body weight gain in %

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable

OPHTHALMOSCOPIC EXAMINATION: Yes, see table 1

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes (identity) Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia
- Animals fasted: Yes, in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: all
- Parameters checked in table No.3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:after 4 weeks
- Animals fasted: Yes, Yes, in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: all
- Parameters checked in table No.4 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.5 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes, see table 1
- Time schedule for examinations: During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.
- Dose groups that were examined: see table 1
- Battery of functions tested: reflexes / grip strength / locomotor activity

OTHER:
Vaginal Smear for Estrus Stage
Vaginal smears were taken over four days from all females, and the stage of estrus was evaluated, if possible (During Week 3 and 4)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see table 2
HISTOPATHOLOGY: Yes, see table 2

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Red stained feces were noted in males and females treated with the test item for four weeks at all dose levels. Otherwise, no test item-related findings were noted at the daily observation. No abnormalities were recorded at the weekly behavioral observations in all animals at any dose level.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In the males treated with 1000 mg/kg/day, a statistically significant decrease in white blood cell (WBC) count was noted in correlation with significantly decreased absolute lymphocyte value.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the males treated with 1000 mg/kg/day, a significant decrease in phosphorus and potassium concentration was noted. Due to the occurrence in the high dose group, these effects are considered to be test item-related, although potassium levels are within the range of historical data. Furthermore, the total protein level was increased in correlation with increased albumin and increased albumin/globulin ratio. As these findings were noted only in the males and there was no dose response relationship, these changes were considered not to be of adverse nature.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No significant differences of absolute organ weights were noted in the males. Significantly increased (p<0.05) liver/body weight ratio was noted in males of all treatment groups compared to control animals. Furthermore, in males treated with 1000 mg/kg/day, the kidney/brain weight ratio was significantly increased (p<0.05). In the females, no significant differences in absolute and relative organ weights were noted. (see Table 1).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Red staining of kidneys and/or lymphnodes and/or stomach was noted in several male and female animals at all dose levels, and is considered not to be indicative of systemic toxicity. Otherwise, no test tem-related findings were noted at necropsy. See Table 2.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Relevant microscopic findings were recorded only in the kidneys. Deposits of a fine granular red pigment were recorded in the tubular epithelium at minimal to slight severity in both sexes treated with 1000 mg/kg/day and in females treated with 300 mg/kg/day. The deposits of red pigment were in some cases associated with increased severity of hyaline droplets in males treated with 1000 mg/kg/day and minimal to slight vacuolar degeneration of tubular epithelium in females treated with 1000 mg/kg/day.

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived the scheduled treatment period.

BODY WEIGHT AND WEIGHT GAIN
No effects on the body weight or body weight gain were noted in male and female animals during four weeks of treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No test item-related difference in the mean daily food consumption was noted at any dose level.

OPHTHALMOSCOPIC EXAMINATION
Daily Observations
Red stained feces were noted in males and females at all dose levels (from day 8 at 100 mg/kg/day, from day 5 at 300 mg/kg/day and from day 3 at 1000 mg/kg/day). Otherwise, no test item-related findings were noted at the daily observation.
In one female of the control group, red scabs around the left eye were noted from day 6 up to the end of treatment. In one female treated with 1000 mg/kg/day, hunched posture and dyspnoe were noted on day 14 of treatment. These findings were considered to be incidental.

Weekly Behavioral Observations
No abnormalities were recorded at the weekly behavioral observations in all animals at any dose level.


HAEMATOLOGY
In the males treated with 1000 mg/kg/day, a distinctive and statistically significant (p<0.01) decrease in white blood cell count (WBC) was noted. This was in correlation with significantly decreased (p<0.01) absolute lymphocyte value. Both values were below the range of historical data.

Further, significant changes in differential WBC count were noted in males treated with 1000 mg/kg/day such as decrease in neutrophils, eosinophils, basophils, monocytes and large unstained cells. However, these values stayed within the historical data even though at the lower limit. Furthermore, in males treated with 300 mg/kg/day, lymphocytes were significantly decreased, but also within the range of historical data for rats of this age, strain and sex. Further, statistically significant changes in males in hemoglobin, hematocrit, mean corpuscular hemoglobin concentration (MCHC) and hemoglobin concentration distribution width (HDW) are all within the historical control data, and, therefore, considered not to be treatment-related. There were no test item-related changes in hematological parameters detected in the females of all dose groups. The few significantly different parameters noted in females treated with 300 mg/kg/day only, were within the historical data range (HDW slightly higher) with no dose response-relationship and, therefore, considered not to be treatment-related.

CLINICAL CHEMISTRY
No adverse test item-related findings in clinical biochemistry parameters were noted. In the males treated with 1000 mg/kg/day, significantly decreased alanine aminotransferase (ALAT) and alkaline phosphatase (ALP) levels were detected. A decrease in these parameters was considered to be within the range of biological variance. In the same animals, a decrease in potassium and phosphorus concentrations was noted. Due to the occurrence in the high dose group, these effects are considered to be test item-related, although potassium levels are within the range of historical data. Furthermore, the total protein level was increased in correlation with increased albumin and increased albumin/globulin ratio. As these findings were noted only in males and as there was no dose response relationship, these changes were considered not to be of adverse nature. There were no changes considered to be test item-related in clinical biochemistry parameters in the females of all dose groups. The few significantly different parameters only in females treated with 300 mg/kg/day, are all within the range of historical data and considered not to be treatment related.

URINALYSIS
No test item-related effects were noted in male and female animals of all dose groups compared to control group.

NEUROBEHAVIOUR
Functional Observational Battery (Screen). No findings were evident during the functional observational battery performed during week 4 of treatment.

Grip Strength
No differences compared to the control group were recorded in the grip strength of male and female animals at any dose level.

Locomotor Activity
There were no effects on the locomotor activity observed in male and female animals of all groups.

ORGAN WEIGHTS (see table 1)
In the males, significantly increased (p<0.05) liver/body weight ratio was noted in all treatment groups. The liver/brain weight ratio as well as the absolute mean liver weight was slightly (nonsignificantly) increased in males of all treatment groups (see table below). The kidney/brain weight ratio was significantly (p<0.05) increased in the males treated with 1000 mg/kg/day. No significant changes were noted in the absolute and relative organ weights of the females.

GROSS PATHOLOGY (see table 2)
Test item-related dark red discoloration of the kidney was noted in four males treated with 300 mg/kg/day and all males treated with 1000 mg/kg/day. In the males treated with 1000 mg/kg/day, the mesenteric lymphnodes showed also a light red discoloration. In the females, dark red discoloration of the kidneys was noted in four out of five animals treated with 100 mg/kg/day and all animals treated with 300 mg/kg/day and 1000 mg/kg/day, respectively. In the females treated at 1000 mg/kg/day, light red discoloration of the lymphnodes was also noted in all animals and discolored (light red) stomach in one animal. Furthermore, pelvic dilation was noted in one control male and red foci in the thymus were recorded in three control males as well as two males treated with 300 mg/kg/day.

HISTOPATHOLOGY: NON-NEOPLASTIC (see table 3)
Relevant microscopic findings were recorded only in the kidneys. The remainder of further findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Kidneys
Deposits of a fine granular red pigment were recorded in the tubular epithelium at minimal to slight severity in both sexes of group 4 and in females of group 3. The deposits of red pigment were sometimes associated with increased severity of hyaline droplets in males of group 4 and minimal to slight vacuolar degeneration of tubular epithelium in females of group 4.

OTHER FINDINGS
Vaginal Smear for Estrus Stage: No special findings were noted concerning the estrous stage of the female animals.

Thyroid Hormone Analysis
No analysis of the thyroid hormone levels was performed, as there were neither alterations in the organ weights nor macroscopic or microscopic changes in the thyroid.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Table 1: Changes in Mean Absolute Organ Weight and Mean Relative Organ Weight

(Organ/Body Weight (bw) or Organ/Brain Weight (brw) Ratio) as Compared to Controls

Organ/ Ratio	Males			Females
	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
Liver weight	+15.3% (-)	+15.4% (-)	+15.8% (-)
Liver/bw ratio	+9.7% (*)	+9.7% (*)	+7.6% (*)
Liver/brw ratio	+16.1% (-)	+14.7% (-)	+14.5% (-)
Kidney weight			+16.1% (-)
Kidney/ bw ratio			+7.4% (-)
Kidney/ brw ratio			+14.6% (*)

*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig.(-)

Table 2:Incidence of Main Macroscopic Findings

Findings/Incidence	Group 1		Group 2		Group 3		Group 4
Terminal Sacrifice	5 M	5 F	5 M	5 F	5 M	5 F	5 M	5 F
Kidneys: Discoloration, red	0	0	0	4	4	5	5	5
Mesenteric lymph node: Discoloration, red	0	0	0	0	0	0	5	5
Stomach: Discoloration, red	0	0	0	0	0	0	0	1

Table 3 Incidence and Mean Severity Grade of Main Findings in Kidneys

Finding	Group 1		Group 2		Group 3		Group 4
Incidence / Mean Severity	5 M	5 F	5 M	5 F	5 M	5 F	5 M	5 F
Deposits, red pigment	-	-	-	-	-	5/1.0	5/1.0	5/1.4
Vacuolar degeneration	-	-	-	-	-	-	-	4/1.8
Hyaline droplets	2/1.0	-	1/1.0	-	2/1.0	-	3/2.0	-

Males Body Weights Summary

	Group 1 0 mg/kg	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4  1000 mg/kg
PRE-RANDOMIZATION
Day 1      MEAN	178	176-	180-	176-
ST.DEV.	7	6	7	6
MINIMUM	169	168	172	166
MAXIMUM	188	183	190	182
N	5	5	5	5
ACCLIMATIZATION
Day 1      MEAN	194	195-	195-	193-
ST.DEV.	6	8	8	6
MINIMUM	186	182	189	187
MAXIMUM	203	205	207	200
N	5	5	5	5
TREATMENT PERIOD
Day 1      MEAN	220	225-	225-	228-
ST.DEV.	9	9	11	5
MINIMUM	205	215	218	222
MAXIMUM	231	239	243	234
N	5	5	5	5
Day 8      MEAN	251	257-	260-	266-
ST.DEV.	13	12	16	10
MINIMUM	232	249	249	253
MAXIMUM	265	278	287	278
N	5	5	5	5
Day 15    MEAN	279	284-	286-	297-
ST.DEV.	17	16	22	12
MINIMUM	255	266	262	283
MAXIMUM	299	308	321	315
N	5	5	5	5
Day 22    MEAN	305	309-	312-	323-
ST.DEV.	20	20	27	16
MINIMUM	273	284	282	309
MAXIMUM	325	340	354	350
N	5	5	5	5
Day 28   MEAN	320	326-	327-	341-
ST.DEV.	22	25	29	21
MINIMUM	283	292	295	323
MAXIMUM	340	361	372	376
N	5	5	5	5

*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig.(-)

Females Body Weights Summary

	Group 1 0 mg/kg	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
PRE-RANDOMIZATION
Day 1      MEAN	145	146-	148-	145-
ST.DEV.	5	5	7	4
MINIMUM	139	140	141	139
MAXIMUM	152	153	159	149
N	5	5	5	5
ACCLIMATIZATION
Day 1      MEAN	156	160-	160-	154-
ST.DEV.	5	2	5	5
MINIMUM	149	158	155	148
MAXIMUM	162	164	168	161
N	5	5	5	5
TREATMENT PERIOD
Day 1      MEAN	169	170-	173-	172-
ST.DEV.	4	5	4	2
MINIMUM	165	161	168	169
MAXIMUM	175	173	178	175
N	5	5	5	5
Day 8      MEAN	182	183-	183-	182-
ST.DEV.	6	3	5	5
MINIMUM	173	178	176	177
MAXIMUM	189	185	189	189
N	5	5	5	5
Day 15    MEAN	191	192-	192-	192-
ST.DEV.	9	8	7	9
MINIMUM	179	181	184	182
MAXIMUM	204	204	199	203
N	5	5	5	5
Day 22    MEAN	201	206-	194-	200-
ST.DEV.	10	8	5	9
MINIMUM	186	196	187	187
MAXIMUM	210	219	200	213
N	5	5	5	5
Day 28   MEAN	207	209-	206-	204-
ST.DEV.	13	7	7	10
MINIMUM	196	198	200	189
MAXIMUM	228	217	215	215
N	5	5	5	5

*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig.(-)

Conclusions:

The no-observed-adverse-effect-level (NOAEL) of FAT 40850/A was established at 300 mg/kg/day.

Executive summary:

The 28-day oral repeat dose study with FAT 40850/A was carried out in Wistar rats according to OECD guideline 407 and EU method B.7 at doses of 100, 300 and 1000 mg/kg/day. The study was performed under GLP-conditions. A control group was treated similarly with the vehicle, bidistilled water, only. Each group comprised 5 animals per sex which were terminated after 28 days of treatment. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization and treatment periods. Functional observational battery, locomotor activity and grip strength were assessed during week 4. At the end of the treatment period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

The following test item related findings were noted:

- Red stained feces, noted in males and females at all dose levels (100, 300 and 1000 mg/kg/day) were considered to be caused by a dyeing effect of the test item but not to be adverse. Red discoloration was also detected macroscopically in multiple organs, like kidneys, mesenteric lymphnodes and stomach.

- A significant decrease in the white blood cell count and in lymphocytes (absolute) with values below ranges of historical control data in males at 1000 mg/kg/day was possibly related to the treatment with the test item. The lymphocyte value in males treated with 300 mg/kg/day was also decreased and there seemed to be a tendency in reduction of lymphocytes (absolute) with an effect on total WBC in the highest dose group. In the absence of histopathologigal changes e.g. in the spleen, bone marrow, thymus or lymphnodes and no findings in the females, this effect is considered not to be adverse.

- A decrease in potassium and phosphorus concentrations was noted in males at 1000 mg/kg/day and, as occurred in the high dose group, is considered to be test item-related, although potassium levels are within the range of historical data. Furthermore, the total protein level was increased in correlation with increased albumin and increased albumin/globulin ratio. As these findings were noted only in males and as there was no dose response relationship, these changes were considered not to be of adverse nature.

- The increase in liver weights is considered to be caused by an adaptive reaction but, in absence of further findings, e.g. pathohistological changes in the liver, not to be of adverse nature.

- Changes in kidney weights in males at 1000 mg/kg/day are in correlation with macroscopic and microscopic findings. Histologically, treatment related findings consisted of minimal to slight deposits of a red pigment in the kidneys of animals of both sexes treated with 1000 mg/kg/day and in females treated with 300 mg/kg/day. The pigment was red fine granular and located in the tubular epithelium, and sometimes associated with increased severity of hyaline droplets in males treated with 1000 mg/kg/day and minimal to slight vacuolar degeneration of tubular epithelium in females of the same dose group. The deposits of red pigment were considered to be not adverse itself, but the secondary reactions consisting of increased severity of hyaline droplets and vacuolar degeneration of tubular epithelium were deemed to be adverse. Based on the results of this study, a no-observed-effect-level (NOEL) of FAT 40850/A could not be established. The no-observed-adverse-effect-level (NOAEL) was established at 300 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality study conducted according to EU Method B.7/ OECD TG 407 and in compliance to GLP

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

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

Based on the findings of repeated dose toxicity study from read-across substance, the Reactive Red 024:1 does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.