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Administrative data

Description of key information

Key information on Reinblau RLW: NOEL = 1000 mg/kg bw/day (OECD 407, GLP, WoE, rel.2)
Key information on Solvent Green 28 (Category member): NOAEL = 1000 mg/kg bw/day (OECD 422, GLP, WoE, rel.1)

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Justification for type of information:
Justification Document for the Category of Six Anthraquinone Dyes

LANXESS Deutschland GmbH has registered five mono-constituent anthraquinone dyes of similar chemical structure using a category approach: Solvent Violet 36 (CAS No 82-16-6), Solvent Green 3 (CAS No 128-80-3), Reinblau RLW (CAS No 41611-76-1), Reinblau BLW (CAS No 32724-62-2) and Solvent Green 28 (CAS No 4851-50-7). Additional data were taken from another registered anthraquinone dye, Solvent Blue 104 (CAS No 116-75-6), leading to a category consisting of six members (see attached justification in the Category dossier).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no signs were observed in association with dose administration.
Clinical signs observed at routine examination comprised several incidences of vocalisation and irritable behaviour in males and females at all dose levels, including Controls. One female receiving 1000 mg/kg/day was recorded to have a twisted muzzle on Day 14 of lactation. No test item-related changes in general clinical condition were observed following treatment with Macrolex Grün G.
Mortality:
no mortality observed
Description (incidence):
There were no premature deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For males receiving Macrolex Grün G, overall mean body weight gains between Weeks 0-5 were marginally low at all dose levels when compared with Control values (control 111 g, dosed 102-105 g). This was primarily due to a slight dose dependent reduction in body weight gain observed in males between Weeks 0-1 following commencement of treatment (control 30 g, 1000 mg/kg 23 g), with statistical significance observed at 1000 mg/kg/day (p=<0.01).

During the two-week pre-pairing treatment period, females receiving 100 mg/kg/day were unaffected by administration with Macrolex Grün G. For females receiving 300 mg/kg/day, a non-significant reduction in body weight gain was observed between Weeks 0-1 (4 g dosed v. 8 g control, 50% lower), and resolved during weeks 1-2 (14 g dosed v. 9 g control, 55% higher). Females given Macrolex Grün G at 1000 mg/kg/day exhibited a greater body weight gain between weeks 0-1, and a decrease in body weight gain between Weeks 1-2 (5 g v. 9 g, 45% lower). Mean body weight gain was unaffected when the whole premating period (Weeks 0-2) was considered.

During the gestation period for females receiving Macrolex Grün G at doses up to and including 1000 mg/kg/day, a dose dependent reduction in overall body weight gains was observed (Days 0-20), although no statistical significance was observed (138 g 1000 mg/kg bw/day v. 154 g control).

Following parturition, body weight performance was similar to that of Controls for females receiving 100, 300 and 1000 mg/kg/day up to Day 13 of lactation.

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption for males receiving Macrolex Grün G at doses up to and including 1000 mg/kg/day was considered to be unaffected by treatment.
For females receiving Macrolex Grün G at doses of 100, 300 and 1000 mg/kg/day, food consumption values were similar to those of the Controls throughout the treatment period, gestation and lactation phases.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The hematological examination of peripheral blood performed after five weeks of treatment for males and on Day 14 of lactation for females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the slightly low neutrophil and eosinophil concentrations in males receiving 100, 300 or 1000 mg/kg/day, low prothrombin times in males at 100, 300 and 1000 mg/kg/day, high platelet count in males at 1000 mg/kg/day and high reticulocyte count in females at 100, 300 and 1000 mg/kg/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical examination of plasma performed after five weeks of treatment for males and on Day 14 of lactation for females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the slightly, but statistically significantly low alkaline phosphatase in males at 300 and 1000 mg/kg/day and alanine aminotransferase in males at 100, 300 and 1000 mg/kg/day and the slightly, but statistically significantly high cholesterol levels in females at 1000 mg/kg/day. Low creatinine concentration was evident in males at 1000 mg/kg/day.
Endocrine findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The evaluation of organ weights of males killed after 5 weeks of treatment and of females killed on Day 13 of lactation revealed marginally low absolute and adjusted thymus weights in females treated at 100 and 1000 mg/kg/day, as well as in males given 1000 mg/kg/day. No microscopic correlates were identified, therefore these differences are of doubtful relationship to treatment. No other treatment-related changes in organ weights were observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic examination performed after 5 weeks of treatment revealed the following changes in the caecum, colon, ileum, jejunum, rectum, stomach and general comments.

Gastrointestinal Tract
Green content, representing the colour of the test item, was seen in the caecum, colon, ileum, jejunum, rectum and stomach in treated animals with a dose dependent increase in incidence.

Stomach
Green colour of the stomach was seen in treated animals with a dose dependent increase in incidence.

General comments
Green staining of the fur was seen in males treated at 1000 mg/kg/day and in one female treated at 100 mg/kg/day.
Green staining of the tail was seen in males treated at 1000 mg/kg/day and females treated at 100, 300 and 1000 mg/kg/day.

Summary tables of the findings can be found in section 'any other information on results'

The incidence and distribution of all other findings were considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment Related Findings
There were no treatment related changes.

Incidental Findings
Mineralisation was seen in the kidneys (cortex, sometimes correlated with macroscopically pale kidneys) and the glandular mucosa of the stomach of control and treated females. In the stomach; minimal mineralisation was seen in controls and treated females at a similar incidence and was considered unrelated to treatment. In the kidneys; histopathological findings associated with the observed distribution of mineralisation included minimal to slight degeneration/necrosis and minimal dilatation of cortical tubules. The incidence and/or severity of the kidney mineralisation and associated findings appeared slightly increased in treated females compared with controls.
A compilation of background control data (BCD) from similar recent studies was performed for the all above findings. In this BCD, mineralisation of the kidney cortex and cortical tubular degeneration/necrosis each have an incidence range of 0 – 100% and a severity range of minimal to moderate. Cortical tubular dilatation has a BCD incidence range of 0 – 83.3%, with a severity range of minimal to moderate. Mineralisation of the glandular stomach mucosa has a BCD incidence range of 0 – 60%, with a severity range of minimal to moderate. All these findings within the current study are well within these ranges.
Therefore, the discussed findings are considered to be incidental, with the apparent slight increase in treated females due to chance and not related to treatment.
The incidence and distribution of all other findings were considered to be unrelated to treatment.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age.

Sensory Reactivity Observations and Grip Strength
The sensory reactivity observations conducted during Week 5 of treatment for males and Day 7-9 of lactation for females revealed no findings which were considered treatment related in animals receiving Macrolex Grün G at doses of 100, 300 and 1000 mg/kg/day.

Motor Activity
The motor activity assessment conducted during Week 5 of treatment revealed no test-item related effects in male or female animals receiving Macrolex Grün G at all dose levels. It was noted that in male animals receiving 1000 mg/kg/day, a lower than Control statistical significance was seen in the low beam break at the 6 minute testing point, as well as a higher than Control statistical significance seen in the low beam break at 60 minutes. These inconsistent, isolated findings were considered not to be associated with administration of Macrolex Grün G.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no

Thyroid Hormone Analysis

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age.

Group

Treatment

Dose

 

Adult Male at Termination

(pg/mL)

Day 13 of age Offspring (pg/mL)

(mg/kg/day)

Male

Female

1

Corn oil

0

Mean

28500

29700

33400

SD

9340

6580

13400

CV

32.8

22.2

40.1

N

10

10

10

2

Macrolex Grün G

100

Mean

27600

37700

37000

SD

11000

7420

5300

CV

39.9

19.7

14.3

N

10

10

10

3

Macrolex Grün G

300

Mean

26700

36000

38500

SD

12500

6540

7660

CV

46.8

18.2

19.9

N

10

10

10

4

Macrolex Grün G

1000

Mean

32000

28800

37400

SD

8570

5390

7890

CV

26.8

18.7

21.1

N

10

10

10

 

Summary of findings in the gastrointestinal tract for animals killed after 5 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

300

500

0

100

300

500

Finding

Abnormal Contents, Green

Caecum

0

3

6

9

0

1

1

4

Colon

0

3

5

9

0

0

1

2

Ileum

0

0

1

2

0

0

1

1

Jejunum

0

0

2

3

0

0

0

0

Rectum

0

0

3

6

0

0

0

2

Stomach

0

1

4

4

0

2

2

6

Number of tissues examined

10

10

10

10

10

10

10

10

Summary of findings in the stomach for animals killed after 5 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

300

1000

0

100

300

1000

Abnormal colour, Green

0

2

5

9

0

8

7

9

Number of tissues examined

10

10

10

10

10

10

10

10

Summary of general comments for animals killed after 5 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

300

1000

0

100

300

1000

Fur stained, Green

0

0

0

8

0

1

0

0

Tail stained, Green

0

0

0

5

0

1

2

6

Number of tissues examined

10

10

10

10

10

10

10

10

Conclusions:
It was concluded that the oral administration of Macrolex Grün G to parental Sprague-dawley rats at dose levels of 100, 300 or 1000 mg/kg/day administered for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation, was well tolerated.
Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, Macrolex Grün G showed no evidence of being an endocrine disruptor.
The no-observed adverse-effect level (NOAEL) of Macrolex Grün G for systemic toxicity was considered to be 1000 mg/kg/day and the no-observed adverse-effect level (NOAEL) of Macrolex Grün G for reproductive/developmental toxicity was also considered to be
1000 mg/kg/day.
Executive summary:

Summary


The purpose of this study was to assess the potential systemic toxic potential in Crl:CD(SD) rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item, Macrolex Grün G, by oral administration for at least five weeks.


Three groups of ten male and ten female rats received Macrolex Grün G at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as the treated groups.


 


During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.


The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. 


 


Results


F0 responses


Administration with Macrolex Grün G at doses up to and including 1000 mg/kg/day was considered to generally be well tolerated throughout the treatment period. There were no premature deaths, no dosing signs and no clinical signs observed in association with dose.


 


For males receiving Macrolex Grün G, overall mean body weight gains between Weeks 0-5 were marginally low at all dose levels when compared with Control values (control 111 g, dosed 102-105 g). This was primarily due to a slight dose dependent reduction in body weight gain observed in males between Weeks 0-1 following commencement of treatment (control 30 g, 1000 mg/kg 23 g), with statistical significance observed at 1000 mg/kg/day (p=<0.01).


 


During the two-week pre-pairing treatment period, females receiving 100 mg/kg/day were unaffected by administration with Macrolex Grün G. For females receiving 300 mg/kg/day, a non-significant reduction in body weight gain was observed between Weeks 0-1 (4 g dosed v. 8 g control, 50% lower), and resolved during weeks 1-2 (14 g dosed v. 9 g control, 55% higher). Females given Macrolex Grün G at 1000 mg/kg/day exhibited a greater body weight gain between weeks 0-1, and a decrease in body weight gain between Weeks 1-2 (5 g v. 9 g, 45% lower). Mean body weight gain was unaffected when the whole premating period (Weeks 0-2) was considered.


 


During the gestation period for females receiving Macrolex Grün G at doses up to and including 1000 mg/kg/day, a dose dependent reduction in overall body weight gains was observed (Days 0-20), although no statistical significance was observed (138 g 1000 mg/kg bw/day v. 154 g control).


 


Following parturition, body weight performance was similar to that of Controls for females receiving 100, 300 and 1000 mg/kg/day up to Day 13 of lactation.


 


Estrous cyclicity, pre-coital interval, gestation length, mating performance, fertility and gestation index were unaffected by treatment.


 


The hematological examination of blood and the biochemical examination of plasma did not reveal any conclusive effect of treatment and there was no effect upon circulating levels of thyroxine (T4) in adult males.


 


Macroscopic findings were limited to green content, representing the colour of the test item, seen in the caecum, colon, ileum, jejunum, rectum and stomach in treated animals with a dose dependent increase in incidence and green colour of the stomach seen in treated animals with a dose dependent increase in incidence. Histopathological findings in the full range of tissues examined were considered to be unrelated to treatment. Organ weights were considered unaffected by treatment.


 


Applicant’s Discussion


A review of findings following conduct of an OECD 422 study with Macrolex Grün G in Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day allows the clear conclusion to be reached of no adverse outomes. For those parameters in which effects were observed, namely bodyweight gain changes, thymus weight, and kidney mineralisation, none may be considered as clearly treatment-related. Bodyweight gain changes were transient in nature and without any clear indications of associated adversity. Changes in the weight of thymus lacked a clear dose-response in female animals, were not significant in either sex, and were without microscopic correlate following histopathological exam. As such, they are not considered to be a treatment-related adverse effect. Furthermore, mineralisation described in the kidney was considered to be incidental following review of historical control data. Finally, the colour changes described during gross pathological evaluation are clearly relatable to the passage of Macrolex Grün G through the gastrointestinal tract, alongside staining of the tail which is very likely to be due to contamination from fecal matter. There is no supporting evidence from this or any other study findings to indicate that the test item was absorbed.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 September 1997 to 18 November 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
When comparing to the OECD TG 407 (1995 version), the following deviations were reported: - sensory reactivity to stimuli and functional observations were not conducted - gross necropsy of testes was not performed - histopathological examinations were not performed on all the organs listed in the OECD TG.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
1995
Deviations:
no
Principles of method if other than guideline:
Reinblau RLW Tr. was administered by oral gavage to male and female SD rats for 28 days at dose levels of 0, 8, 40, 200, and 1000 mg/kg to assess its toxicity and reversibility.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: n/a
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: the stability of the lot used in this study was confirmed but the Sponsor using IR analysis
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: n/a
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: The stability and homogeneity of the test substance in the dosing formulations were confirmed between 0.4 and 100 mg/mL for 8 days in a refrigerator before the start of dosing.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): n/a
- Preliminary purification step (if any): n/a
- Final concentration of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a
Species:
rat
Strain:
Crj: CD(SD)
Remarks:
Crj: CD(SD) IGS
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles Liver Laboratories Japan Ltd. on September 24, 1997
- Females (if applicable) nulliparous and non-pregnant: yes; 48 females; 48 males.
- Age at study initiation: 5 weeks
- Weight at study initiation:146 g to 184 g for males and 119 g to 146 g for females.
- Fasting period before study: none
- Housing: polycarbonate cages (265W x 426D x 200H mm, Tokiwa Kagaku Kikai Co., Ltd.) with sterilized hardwood chip for experimental animals (Beta chip, Charles River Laboratories Japan Ltd.). After grouping, two animals were housed in a cage, which was placed on stainless steels racks (Tokiwa Kagaku Kikai Co., Ltd.), and the location of the cages was rotated once a week. Replaced once a week
- Diet (e.g. ad libitum): pellet diet ad libitum (MF, Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): ad libitum, tap water passed through 5 µm filter and irradiated with UV light. Replaced once a week.
- Acclimation period: 13 days.

DETAILS OF FOOD AND WATER QUALITY: it was confirmed that the contaminants such as residual pesticides in the diet and bedding were meet the specifications of the standard operating procedure (SOP) of the test facility. In addition, the tap water was periodically analyzed based on the Water Supply Act, and was confirmed to meet the specification of the SOP of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C (intended value)
- Humidity (%): 55±15%(intended value)
- Air changes (per hr):ventilation was about 12 times per hour
- Photoperiod (hrs dark / hrs light):12/12 (light from 7:00 to 19:00).

IN-LIFE DATES: From: 24 September 1997 To: 4 November 1997.
Route of administration:
oral: gavage
Details on route of administration:
Oral route was selected according to the guideline.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
CMC-Na solutions
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing volume was set at 10 mL/kg, and the individual dose volume was calculated on the basis of the most recent body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: 0.5% (test item suspended with motor and agate)
- Amount of vehicle (if gavage): n/a
- Lot/batch no. (if required): Iwai Chemicals Company Ltd.; Lot Nos. 121208 and 100709
- Purity:n/a
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity for 8 days of the test substance in the dosing formulations at 0.4 and 100 mg/mL were confirmed by the HPLC method.
The dosing formulations of initial preparation were analyzed and confirmed that the mean concentrations were within the nominal concentration ±10%.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily in the morning
Dose / conc.:
8 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control + 200 + 1000 mg/kg bw/day: 12 / sex (incl. 6/sex recovery animals).
8 and 40 mg/kg bw/day: 6 / sex.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: From the results of a preliminary study, the highest dose in this study was set at 1000 mg/kg, and then, doses of 200, 40, and 8 mg/kg were also set by common ratio of 4.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: no
- Rationale for selecting satellite groups: control and high dose groups
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): not applicable
- Dose range finding studies: In a preliminary 2-week repeated dose study (dose level: 0, 100, 500, and 1000 mg/kg bw/day; 3 males and 3 females per group), there were no changes attributable to the test substance treatment in clinical sign, body weight, haematology, organ weight, and necropsy.
- Other: none
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day (before and after dosing) during the dosing period, and once in the morning during the other periods.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
-All feeders in each cages (including diet) were weighed using an electric balance once a week, and mean daily food consumption of each animal was calculated.

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: Not specified

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the scheduled necropsy day (days 29 or 43).
- Anaesthetic used for blood collection: Yes (sodium thiopental)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [7.5.1/1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the scheduled necropsy day (days 29 or 43).
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [7.5.1/1] were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: day 24
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: No
- Parameters checked in table [7.5.1/1] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: n/a
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.1/2)

HISTOPATHOLOGY: Yes (see table 7.5.1/2)
At the results of the examination, since there were no changes suspected to be treatment-related, histopathological examination was not performed in the other groups necropsied at the end of the treatment period and all groups necropsied at the end of the recovery period.
Optional endpoint(s):
Optional endpoints: No
Statistics:
Numerical data were analyzed statistically by the multiple comparison tests.
Numerical data were analyzed statistically by the multiple comparison tests. First, the data were tested by Bartlett’s test for homogeneity of variance. When the variance was homogeneous, one-way analysis of variance (ANOVA) was employed, and when the variance was heterogeneous, the Kruskal-Wallis test was performed. When there was a significant difference among the groups, the Dunnett test or the Dunnett-type multiple comparison test was applied. Categorical data were tested by X test (a*b). When there was a significant difference among the groups, Armitage’s X test was applied to compare between the control group and each dose group.
Statistical analysis was performed on the items listed below. The analysis was not performed on the data obtained in the clinical observation, necropsy, and histopathological examination.
Multiple comparison tests: Xtest:
Body weight, food consumption, hematology, blood chemistry, organ weight
Urinalysis (pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen)
Clinical signs:
no effects observed
Description (incidence and severity):
There were no abnormalities in any groups including the control group.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight of animals in the test substance-treated groups was comparable to that in the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of animals in the test substance-treated groups was comparable to that in the control group.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no abnormalities attributable to the test substance treatment at the end of the treatment or recovery periods.
Low values of hemoglobin concentration and hematocrit value were noted in males in the 200 mg/kg bw/day group, and shortening of APTT was noted in females in the 200 mg/kg bw/day group at the end of the recovery period; however, these changes were not considered to be treatment-related because similar changes were not noted at the end of the treatment period or in the 1000 mg/kg bw/day group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no abnormalities attributable to the test substance treatment at the end of the treatment or recovery periods.
A high value of total bilirubin was noted in females in the 200 mg/kg bw/day group; however, similar change was not noted in the 1000 mg/kg bw/day group, and therefore, this change was not considered to be treatment-related. Moreover, low vales of albumin and sodium were noted in males in the 1000 mg/kg bw/daygroup, and a high value of potassium was noted in males in the 200 and 1000 mg/kg bw/day groups; however, these changes were not considered to be treatment-related because these were slight and within the physiological range, and were not noted at the end of the treatment period.
Endocrine findings:
not examined
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no abnormalities attributable to the test substance treatment at the end of the treatment period.
A high value of protein was noted in males in the 8, 40, and 1000 mg/kg bw/day groups; however it was not considered to be treatment-related because it was within the physiological range.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no significant differences between the control and test substance-treated groups in any organs.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no abnormalities attributable to the test substance treatment.
The following changes were noted; however, since these changes were observed spontaneously in this strain, they were not considered to be treatment-related: erythrocytic extramedullary hematopoiesis in the spleen, eosinophilic inclusion body in the hepatocyte, fatty change of the periportal hepatocyte, microgranuloma and focal necrosis in the liver, cyst and hyaline droplet in the proximal tubular epithelium in the kidney.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no abnormalities attributable to the test substance treatment. Hepatodiaphragmatic nodule in the liver and cyst in the kidney were noted in the test substance-treated group; however, these changes were not considered to be treatment-related because these were often observed as spontaneous changes in this strain, and there was no dose-relation.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
serum/plasma biochemistry
Key result
Critical effects observed:
no
Conclusions:
The no-observed-effect-level (NOEL) of Reinblau RLW was considered to be 1000 mg/kg bw/day for male and female rats.
Executive summary:

In a subacute toxicity study performed similarly to OECD TG 407 and in compliance with GLP, Reinblau RLW suspended in CMC-Na solutions was administered to Crl: CD SD IGS rats (6/sex/dose in the control and high dose groups; 6/sex/dose in the other groups) by gavage at dose levels of 0, 8, 40, 200 and 1000 mg/kg bw/day. In addition, 6 rats/sex/dose in the control and 1000 mg/kg bw/day groups were subjected to a recovery period of 14 days.


 


There were no test substance-related toxic changes in clinical sign, body weight, food consumption, hematology, blood chemistry, urinalysis, organ weight, necropsy, or histopathology.


Therefore, the no-observed-effect-level (NOEL) of Reinblau RLW was considered to be 1000 mg/kg bw/day for male and female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study on Reinblau RLW is GLP-compliant and of good quality (Klimisch score = 2). However, as some deviations were noted compared to the OECD TG 407, a weight-of-evidence using data on Solvent Green 28 - another member of the category - was preferred to fulfil this endpoint. The study on Solvent Green 28 is GLP-compliant and of high quality (Klimisch score = 1).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Studies on Reinblau RLW and category members are summarized below.


 


1/ Reinblau RLW


In a subacute toxicity study performed similarly to OECD TG 407 and in compliance with GLP, Reinblau RLW suspended in CMC-Na solutions was administered to Crl: CD SD IGS rats (6/sex/dose in the control and high dose groups; 6/sex/dose in the other groups) by oral gavage at dose levels of 0, 8, 40, 200 and 1000 mg/kg bw/day. In addition, 6 rats/sex/dose in the control and 1000 mg/kg bw/day groups were subjected to a recovery period of 14 days.


There were no test substance-related toxic changes in clinical observations, body weight, food consumption, hematology, blood chemistry, urinalysis, organ weight, necropsy, or histopathology.


Therefore, the no-observed-effect-level (NOEL) of Reinblau RLW was considered to be 1000 mg/kg bw/day for male and female rats.


 


2/ Solvent Green 28


In an OECD guideline 422 study (Combined Repeated Dose Toxicity Study and Reproductive/ Developmental Toxicity Screening Study in the Crl:CD(SD) Rat by Oral Gavage Administration) Solvent Green 28 was administered to three groups of ten male and ten female rats by oral gavage administration, for approximately 6 weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day.  A similarly constituted control group received the vehicle, corn oil, at the same volume dose as the treated groups.


The no-observed adverse-effect level (NOAEL) of Solvent Green 28 for systemic toxicity was considered to be 1000 mg/kg/day and the no-observed adverse-effect level (NOAEL) of Solvent Green 28 for reproductive/developmental toxicity was also considered to be 1000 mg/kg/day.


 


3/ Solvent Blue 104 (Source: ECHA disseminated dossier)


Oral administration to Wistar rats at doses of 100, 300 and 1000 mg/kg/day, for 28 days resulted in no substance related deaths, no clinical symptoms (at daily or weekly observations), no clinical symptoms (at the functional observational battery), no effects on food consumption or body weight development, no effects upon hematology and clinical biochemistry parameters, no changes in mean absolute or relative organ weights, and no macroscopical or microscopical findings of toxicological relevance.
Based on the results of this study, 1000 mg/kg body weight/day was established as the no-observed-adverse-effect-level (NOAEL) for the test substance.


 


Conclusion:





The available three short term studies consistently show no toxicity of the anthraquinone dye group; up to the limit dose no adverse effects are reported, and it can be concluded that “no hazard” is identified in any of these studies.




Justification for classification or non-classification

Harmonised classification:


The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 


 


Self-classification:


Based on the available information, no self-classification is proposed according to the CLP or GHS.