1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

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).

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone

EC Number:

255-460-7

EC Name:

1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone

Cas Number:

41611-76-1

Molecular formula:

C32H30N2O2

IUPAC Name:

1,4-bis[(2-ethyl-6-methylphenyl)amino]-9,10-dihydroanthracene-9,10-dione

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths.

Body weight and weight changes:

effects observed, 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. 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, with statistical significance observed at 1000 mg/kg/day.
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 reduction in body weight gain was observed between Weeks 0-1 (50% lower than Control). Females given Macrolex Grün G at 1000 mg/kg/day exhibited a similar decrease in body weight gain between Weeks 1-2. 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), a lthough no statistical significance was observed.
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, treatment-related

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.

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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.

All females showed diestrus at termination.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Estrous cyclicity, pre-coital interval, gestation length, mating performance, fertility and gestation index were considered unaffected by treatment with Macrolex Grün G.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

(Reproductive toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No signs were recorded that were considered to be related to parental treatment with Macrolex Grün G.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

All females that mated were pregnant.
Litter size, offspring survival to Day 13 of age and sex ratio were unaffected by parental treatment with Macrolex Grün G.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight performance of both male and female offspring up to Day 13 of age remained similar to those of the Controls at all dose levels. Thus, there were no treatment-related effects on body weights of offspring of parental animals given Macrolex Grün G at doses of 100, 300 or 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination of offspring revealed no test item related lesions.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Offspring Ano-genital Distance
Mean ano-genital distances in males and females were considered to be unaffected by treatment.

Thyroid Hormone Analysis
There was considered to be no effect of treatment upon T4 levels in offspring on Day 13 of age.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

clinical biochemistry

gross pathology

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive 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 forat least five weeks.

Three groups of ten male and ten female rats receivedMacrolex Grün Gat 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 administration.

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. 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, with statistical significance observed at 1000 mg/kg/day.

During the two-week pre-pairing treatment period, females receiving 100 mg/kg/day were unaffected by administration withMacrolex Grün G. For females receiving 300 mg/kg/day, a reduction in body weight gain was observed between Weeks 0-1 (50% lower than Control). Females given Macrolex Grün G at 1000 mg/kg/day exhibited a similar decrease in body weight gain between Weeks 1-2.Mean body weight gain wasunaffectedwhen 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. 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 dosedependent increase in incidence and greencolour 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.

F1 responses

The clinical condition, litter size, sex ratio, survival indices and body weight and weight gain of offspring were unaffected by parental treatment.

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

The ano-gential distances of offspring were unaffected by paternal treatment and no nipples were seen on any male offspring on Day 13 of age.

No macroscopic findings considered to be related to paternal treatment were recorded.

 

Conclusion

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 administeredfor 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 Gshowed 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.