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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

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).

Data source

Materials and methods

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-bis[(2,6-diethyl-4-methylphenyl)amino]anthraquinone
EC Number:
251-178-3
EC Name:
1,4-bis[(2,6-diethyl-4-methylphenyl)amino]anthraquinone
Cas Number:
32724-62-2
Molecular formula:
C36H38N2O2
IUPAC Name:
1,4-bis[(2,6-diethyl-4-methylphenyl)amino]-9,10-dihydroanthracene-9,10-dione

Test animals

Strain:
other:

Results and discussion

Results of examinations

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.
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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.